Tài liệu Báo cáo khoa học: Multiple enzymic activities of human milk lactoferrin ppt

Multiple enzymic activities of human milk lactoferrin
Tat’yana G. Kanyshkova
1
, Svetlana E. Babina
2
, Dmitry V. Semenov
1
, Natal’ya Isaeva
3
,
Alexander V. Vlassov
1
, Kirill N. Neustroev
4
, Anna A. Kul’minskaya
4
, Valentina N. Buneva
1
and Georgy A. Nevinsky
1
1
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia;
2
Novosibirsk State University, Novosibirsk, Russia;
3
Institute of Cytology and Genetics, Siberian Division of the
Russian Academy of Sciences, Novosibirsk, Russia;
4
Petersburg Nuclear Physics Institute of Russian Academy of Sciences,
St Peterburg, Russia
Lactoferrin (LF) is a Fe

3+
-binding glycoprotein, first
recognized in milk and then in other human epithelial
secretions and barrier fluids. Many different functions have
been attributed to LF, including protection from iron-
induced lipid peroxidation, immunomodulation and cell
growth regulation, DNA binding, and transcriptional acti-
vation. Its physiological role is still unclear, but it has been
suggested to be responsible for primary defense against
microbial and viral infection. We present evidence that
different subfractions of purified human milk LF possess
five different enzyme activities: DNase, RNase, ATPase,
phosphatase, and malto-oligosaccharide hydrolysis. LF is
the predominant source of these activities in human milk.
Some of its catalytically active subfractions are cytotoxic and
induce apoptosis. The discovery that LF possesses these
activities may help to elucidate its many physiological
functions, including its protective role against microbial and
viral infection.
Keywords: enzymic activities; human milk; lactoferrin;
protection.
Lactoferrin (LF) is a single polypeptide chain of 76–80 kDa,
containing two lobes [1], each of which binds one Fe
3+
ion
and contains one glycan chain [2]. It was first recognized
in milk and then in other human epithelial secretions and
barrier body fluids [3–6]. Many different functions have
been attributed to LF, including protection from iron-
induced lipid peroxidation, immunomodulation and cell

growth regulation [6,7], DNA binding [6], RNA hydrolysis
[8,9], and transcriptional activation of specific DNA
sequences [10,11]. It is a potent activator of natural killer
cells [12] and may have an antitumor role [7,13], an activity
that is independent of iron. LF also influences granulo-
poiesis [14], antibody-dependent cytotoxicity [15], cytokine
production [16], and growth of some cells in vitro [17]. The
physiological role of LF and the mechanisms underlying
these activities are still unclear, but it has been suggested to
be responsible for primary defense against microbial and
viral infection [3,5]. LF is a protein of the acute phase; the
highest concentration is usually detected in the inflamma-
tory nidus. It is detected in the blood of newborn babies
several hours after feeding, and can readily penetrate any
cell and nuclear membrane [18]. Owing to its antiviral and
antimicrobial activities, LF increases the passive immunity
of newborns. It was initially suggested that the antimicro-
bial properties of LF may be attributed to its iron-binding
capacity; removal of iron from the microbial environment
is an important defense mechanism as it is needed for the
proliferation of microflora [19]. Many micro-organisms
express surface receptors for LF and it may show different
iron-independent antimicrobial and antiviral properties
[20,21], the mechanisms of which are still a matter of
debate.
We have proposed that, as LF is a relatively small
protein, its polyfunctional properties may result from its
existence in several oligomeric forms that have different
activities, and that its oligomerization and dissociation are
under the control of specific ligands such as ATP [22,23].

In support of this idea, we have shown recently that LF
possesses an ATP-binding site and that interaction of the
protein with ATP leads to changes in its interaction with
polysaccharides, DNA and proteins [23]. We have further
demonstrated that LF possesses two DNA-binding sites,
which interact with specific and nonspecific DNAs in an
antico-operative manner and may coincide or overlap with
the known polyanion-binding and antimicrobial domains of
the protein [24].
Here we show that this extremely polyfunctional protein
possesses five enzyme activities (DNase, RNase, ATPase,
phosphatase, and malto-oligosaccharide hydrolysis). The
RNA-hydrolyzing and DNA-hydrolyzing subfractions of
LF may contribute to its protective role through hydrolysis
of viral and bacterial nucleic acids. In addition, we show
that some catalytic forms of LF are cytotoxic and
Correspondence to G. A. Nevinsky, Laboratory of Repair Enzymes,
Novosibirsk Institute of Bioorganic Chemistry, 8, Lavrentieva Ave.,
630090, Novosibirsk, Russia.
Fax: 007 3832 333677, Tel.: 007 3832 396226,
E-mail:
Abbreviations: LF, human milk lactoferrin; EPS, 4-nitrophenyl
4,6-O-ethylidene-a-
D
-maltoheptaoside.
(Received 23 January 2003, revised 13 May 2003,
accepted 11 June 2003)
Eur. J. Biochem. 270, 3353–3361 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03715.x
apoptosis-inducing agents. These findings suggest that LF
of milk and other human epithelial secretions and body

fluids may contribute to cell defense by policing the function
of human cells.
Materials and methods
Materials and chemicals
Reagents were obtained mainly from Sigma and Merck.
4-Nitrophenyl 4,6-O-ethylidene-a-
D
-maltoheptaoside (EPS)
was purchased from Boehringer Mannheim (Germany).
We also used heparin, antibodies to human LF (Sigma),
DEAE-cellulose DE-52 (Whatman), heparin–Sepharose
and Cibacron Blue–Sepharose CL-6b (Pharmacia Fine
Chemicals), and Toyopearl HW-55 fine (Toyo Soda).
Radioisotopes were purchased from Amersham
(3000 CiÆmmol
)1
).
Purification and analysis of LF
LF was purified and analyzed individually from the milk
of each of 30 donors. Electrophoretically and immuno-
logically homogeneous LF was obtained by sequential
chromatography of human milk proteins on DEAE-
cellulose, heparin–Sepharose, and anti-LF–Sepharose col-
umns [23,24]. It was further chromatographed on a
Cibacron Blue–Sepharose column (15 · 5 mm) as des-
cribed previously [9] with the following modifications: the
column was equilibrated in 20 m
M
sodium acetate, and
LF (pH 4.0) was loaded and eluted with 50 m

M
Tris/HCl
buffer, pH 7.5, and then with a concentration gradient of
NaCl (0–1
M
) in the same buffer. Fractions were collected,
dialyzed at 4 °C for 12 h against 10 m
M
Tris/HCl,
pH 7.5, and their enzyme activities measured (see below).
The N-terminal amino-acid sequences of five subfractions
of LF (see Fig. 2) were determined by the phenyl
isothiocyanate procedure using a liquid-chromatography
system (Hewlett-Packard Co.). SDS/PAGE and immuno-
blotting analysis were performed as described previously
[23,24]. Gel filtration of LF after pH shock was performed
as in [25]. Synthesis of 2¢,3¢-dialdehyde derivatives of
substrates and affinity labeling of LF was carried as
described previously [23,24].
Nucleic acid-hydrolyzing and phosphatase activity of LF
DNA-hydrolyzing activity was assayed in a mixture (20 lL)
containing 150 ng supercoiled pBR322 DNA or phage k
DNA, 5.0 m
M
MgCl
2
,1.0m
M
EDTA, 20 m
M

Tris/HCl
buffer, pH 7.5, and 0.1–1.0 l
M
LF incubated for 1–2 h at
37 °C. The cleavage products were analyzed by electro-
phoresis on a 1.0% agarose gel and ethidium bromide
staining; gels were photographed and the films scanned to
calculate relative activities.
For evaluation of the nuclease activities, various
5¢-[
32
P]ribo-oligonucleotides and deoxyribo-oligonucleo-
tides (1–10 l
M
) were incubated with 0.1–1.0 l
M
LF in
10–20 lL reaction mixture containing 20 m
M
Tris/HCl,
pH 7.5, and 1.0 m
M
EDTA with or without 5.0 m
M
MgCl
2
for 2–6 h at 37 °C, and the products were analyzed in a
20% polyacrylamide gel containing 8
M
urea.

Phosphatase activity was assayed under the same condi-
tions: removal of [
32
P]P
i
from 5¢-[
32
P]oligonucleotides was
assayed by TLC in dioxane/NH
4
OH/water (5 : 1 : 4, by
vol.) on Kieselgel plates (Merck). After chromatography,
theplatesweredried,the[
32
P]products localized by
autoradiography, and their radioactivity was measured by
Cherenkov counting.
The same conditions were used to study cleavage of
human tRNA
Phe
prepared as described previously [26,27]
and labeled at the 5¢ end [26,27]. tRNA (0.1 lgÆmL
)1
;10
5
Cherenkov counts per sample) were incubated at 37 °Cfor
30 min with LF (0.1–1.0 l
M
)orRNaseA(5· 10
)5

mgÆmL
)1
), and the products were analyzed by electropho-
resis in 15% polyacrylamide/8
M
urea gels, with partial
RNase T1 and imidazole digests of the tRNAs run in
parallel to identify the products [27]. Quantification was
performed by analysis on a Fujix BioImaging Analyzer
BAS 2000 System (Fuji).
Nucleotide-hydrolyzing activity
Reaction mixtures (10–20 lL) contained optimal concen-
trations of the standard components (1.0 m
M
MgCl
2
,
0.3 m
M
EDTA, 50 m
M
Tris/HCl, pH 6.8, 100 m
M
NaCl),
0.05–0.2 mgÆmL
)1
LF, and different concentrations of
[c-
32
P]ATP, and were incubated for 0.5–6 h at 37 °C. For

screening column fractions during purification of IgG,
2–3 lL of each fraction was incubated in 10 lL standard
reaction mixture containing 0.1 m
M
[c-
32
P]ATP or [a-
32
P]
ATP (10
7
c.p.m.). The products of nucleotide hydrolysis
were analyzed by TLC in 0.25
M
KH
2
PO
4
buffer, pH 7.0,
on polyethyleneimine–cellulose plates (Merck); the plates
were dried, and the positions of various
32
P-labeled products
were identified using [
32
P]nucleotide standards and auto-
radiography. The radioactivity of the regions corresponding
to P
i
and different nucleotides was measured by Cherenkov

counting.
Amylase activity of LF
Reaction mixtures containing 30 m
M
Tris/HCl, pH 7.5,
1m
M
NaN
3
,1–5m
M
oligosaccharide, and 1–10 l
M
LF
were incubated at 37 °C. The products of hydrolysis of EPS
(12 mgÆmL
)1
) and eight other oligosaccharides were iden-
tified using TLC on Kieselgel 60 plates (Merck; ethanol/
butanol/water, 2 : 2 : 1, by vol.). The plates were dried,
sprayed with 5% H
2
SO
4
in propan-2-ol and again dried at
110 °C to visualize the carbohydrates as described in [28].
Specificity of the hydrolysis of malto-oligosaccharides was
determined after separation of products by TLC and HPLC
on a Lichrosorb-NH
2

column [28]. One unit of activity was
defined as the quantity of LF that released 1 lmolÆL
)1
reducing sugar from maltoheptaose per min at 37 °C,
similar to known amylases [28].
In situ
gel assay of enzymic activities
Enzymic activities of LF were determined in situ by SDS/
PAGE (12% gels). To detect RNase and DNase activities,
gels contained 200 lgÆmL
)1
yeast total RNA or 20 lgÆmL
)1
calf thymus DNA [25,29–31] added to the gel solution
before polymerization. After electrophoresis, the gel was
3354 T. G. Kanyshkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003
washed with a solution of 4
M
urea and twice with water to
remove SDS, and then to allow protein renaturation it was
incubated for 16 h at 37 °Cin20m
M
Tris/HCl buffer,
pH 7.5, containing 1 m
M
EDTA and 5.0 m
M
MgCl
2
.To

reveal the regions of DNA or RNA hydrolysis, the gel was
stained with ethidium bromide. Proteins were revealed by
Coomassie R250 staining.
ATPase was detected using our modification of the
Gomori method for histochemical determination of ATP-
ases [32]. After electrophoresis, SDS was removed by
incubating the gel for 30 min at 37 °C with water (5 times)
and then with 0.5
M
sodium acetate, pH 6.8 (3 times). To
allow protein renaturation and to detect P
i
resulting from
ATP hydrolysis, the gel was incubated for 12 h at 37 °Cin
5m
M
sodium acetate (pH 6.8) containing 1.0 m
M
MgCl
2
,
3m
M
Pb(NO
3
)
2
, and 100 lCi [c-
32
P]ATP. Nonspecifically

adsorbed Pb(NO
3
)
2
was removed by washing the gel 3 times
(10 min) with water, then with hot 5% acetic acid and again
with water. The gel was autoradiographed to detect [
32
P]P
i
.
Phosphatase and amylase activity was determined using
gels without substrates. After electrophoresis, SDS was
removed by incubating the gels as for analysis of DNase
activity. The gels were then cut into 2 mm slices which were
incubated with 20 m
M
Tris/HCl, pH 7.5, at 4 °C for 12 h.
The gel slices were removed by centrifugation, and amylase
or phosphatase activity was assayed using 5¢-[
32
P](pT)
8
or
EPS as described above.
In situ gel assays of the enzymic activities of human milk
proteins (3–7 lL dialyzed human plasma) and the limited
proteolytic cleavage products of LF (2–7 lg) were as
described above for purified LF. Partial proteolytic cleavage
of LF was performed using 0.1–0.5% trypsin (w/w of LF) in

0.1
M
Tris/HCl (pH 8.2)/25 m
M
CaCl
2
at 37 °Cfor4h[33].
K
m
and V
max
for the hydrolysis of different substrates
were determined by the method of initial rates using
nonlinear regression analysis. Errors in the values were
within 10–30%.
Cytotoxicity assays
Tumor cell lines L929 (mouse fibroblasts) and HL-60
(human promyelocytes) were cultured at 37 °Cin0.1mL
Dulbecco’s modified Eagle’s medium containing 5% fetal
bovine serum to confluence. They were then treated with
mitomycin (1 mgÆmL
)1
) for 5 h and washed with medium.
Fresh medium containing different concentrations
(10–100 n
M
) of subfractions of LF-1 to LF-5 (see below)
or tumor necrosis factor (10 n
M
) was then added. The cells

were cultivated for a further 12–48 h, and the percentage of
dead cells, counted after staining with trypan blue every
3–12 h, was compared with that in a control culture. The
results are mean ± SD from at least three different
experiments using three preparations of one to five fractions
of LF (see below) from different milk donors.
DNA fragmentation and annexin V staining
of apoptotic cells
Cells were incubated with LF subfractions (10–100 n
M
)as
described above for 12–24 h, lysed, centrifuged at 20 000 g,
and the supernatant was extracted with phenol/chloroform.
DNA fragments were electrophoresed in a 1.2% agarose gel
and visualized with ethidium bromide [34]. An Annexin-V-
Fluorescein kit was used for analysis of apoptosis according
to instructions provided by the manufacturer (Boehringer-
Mannheim).
Results
Purification and characterization of LF subfractions
We isolated and analyzed separately LF preparations from
the milk of 30 different healthy mothers. LF was purified
from the fraction of human milk that was not adsorbed by
DEAE-cellulose by chromatography on heparin–Sepharose
[22–24], and electrophoretically homogeneous LF was
purified on anti-LF–Sepharose (Fig. 1). As shown previ-
ously [9], human milk LF could be separated into several
distinct isoforms by affinity chromatography on Cibacron
Blue–Sepharose. We found that chromatographically,
electrophoretically, and immunologically homogeneous

LF (after anti-LF–Sepharose chromatography) contains
subfractions with different affinities for Cibacron Blue–
Sepharose (Fig. 2A–C). They all possessed the N-terminal
amino-acid sequence reported for LF, Gly-Arg-Arg-Arg-
Arg-Ser-Val-Glu [9], and also a product of partial proteo-
lytic cleavage [23,24].
Four prominent protein peaks corresponding to LF were
eluted from Cibacron Blue–Sepharose (Fig. 2). The main
subfraction of LF (peak 4, Fig. 2) had the highest affinity
for this sorbent. Three additional subfractions (peaks 1–3,
Fig. 2) represented  10–20% of the total LF dependent on
the milk donor. The first protein peak showed no enzyme
activity, but the three other peaks showed oligonucleotide
5¢-phosphatase, DNase, RNase, ATPase, and malto-oligo-
saccharide-hydrolyzing activities, each activity being eluted
in several peaks (Fig. 2A–C). The LF subfraction corres-
ponding to peak 2 possessed four different activities:
phosphatase, DNase, RNase, ATPase. Eluate correspond-
ing to protein peak 3 showed three prominent peaks of
oligonucleotide 5¢-phosphatase activity (Fig. 2B) and two
peaks of RNase activity (Fig. 2C). Interestingly, two
Fig. 1. Chromatography of LF on anti-LF–Sepharose. Solid line, A
280
;
symbols, activity as percentage of the fraction with maximal activity.
Aliquots (1–3 lL) of column fractions were incubated with phage k
DNA (7.5 lgÆmL
)1
), 5¢-[
32

P](pU)
10
or 5¢-[
32
P](pT)
8
(5 l
M
), [c-
32
P]ATP
(0.5 l
M
), or EPS (12 mgÆmL
)1
)at37°C for 1–4 h. The details of the
experiment are given in Materials and methods.
Ó FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3355
additional DNase peaks were revealed in fractions 15–30
(position of peak 3), but profiles of these activity peaks did
not correlate with that for protein peak 3, with the second
and third DNase peaks occurring between protein peaks 2
and 3 and protein peaks 3 and 4, respectively (Fig. 2A).
There was good correlation between the positions of two
peaks of malto-oligosaccharide-hydrolyzing activity, and
protein peaks 3 and 4 (Fig. 2C). Taking all the data
together, we divided the LF subfractions possessing differ-
ent activities into five subfractions (LF-1 to LF-5) as shown
in Fig. 2. The data on the relative activities of LF-1 to LF-5
in the different enzymatic reactions are collected in Table 1.

The samples of all 30 donors of LF not fractionated on
Cibacron Blue–Sepharose had detectable levels of all five
activities, but these activities were remarkably dependent on
the donor. LF preparations from seven different donors
were analyzed in more detail after fractionation of LF
subfractions on Cibacron Blue–Sepharose. Table 1 shows
the range of variation in the relative activities of the
subfractions depending on the milk donor.
Interestingly, the phosphatase activity of LF varied more
than other activities when compared with the DNAse
activity. Phosphatase activity varied between 20% and 80%
of the DNAse activity for different donors.
Catalytic activities of LF
Five enzyme activities were ascribed specifically to LF, as
shown by several different methods developed in our
laboratory to study the enzyme activities of catalytic
antibodies [29,30,35,36]. Chromatography of purified (but
not fractionated on Cibacron Blue–Sepharose) LF on
Sepharose bearing immobilized antibodies to LF led to
essentially complete binding of LF to the sorbent (Fig. 1).
During protein elution from this column with an acidic
buffer, pH 2.6, the five activities analyzed coincided exactly
with the LF peak, and there were no other peaks of activity.
The same result was obtained with the separated protein
subfractions LF-1 to LF-5. In addition, incubation all five
enzyme peaks corresponding to the subfractions (Fig. 2)
with immobilized LF antibodies led to essentially complete
binding of LF to the sorbent and disappearance of all five
enzyme activities from the solution. All of the enzyme
activities were suppressed by addition of polyclonal LF

antibodies to the reaction mixtures (data not shown).
Usually strong noncovalent protein complexes dissociate
under acidic conditions. To ensure that other proteins were
not tightly bound to purified LF, the combined fractions
from Cibacron Blue–Sepharose (fractions 5–33, Fig. 2) were
incubated at pH 2.4, which usually dissociates strong
noncovalent complexes. They were then repurified by gel
filtration. A single peak corresponding to LF was recovered
(see Fig. 4A), which contained 80–95% of all five enzyme
activities loaded on the column. There were no other peaks
of activity or protein. The same result was obtained for
separated subfractions LF-1, LF-3 and LF-5 (Fig. 2),
corresponding to LF from the milk of three different
donors (data not shown).
Affinity labeling of enzymes with
32
P analogs of their
specific ligands is the most sensitive method for revealing
any contaminating proteins interacting with the same
ligands. As we showed previously, LF possesses an ATP-
binding site, which became labeled after incubation with an
affinity probe for ATP-binding sites, the 2¢,3¢-dialdehyde
derivative of ATP (oxATP), with a stoichiometry of 1.0 mol
[a-
32
P]oxATP bound per mol LF [23]. In addition, LF
Fig. 2. Chromatography of LF on Cibacron Blue–Sepharose. (A)
DNAse (d) and ATPase (s); (B) 5¢-oligonucleotide phosphatase (n);
(C) RNase (*) and amylase (j). Aliquots (1–3 lL) of column fractions
were used to determine DNAse (k DNA), RNase {5¢-[

32
P](pU)
10
},
phosphatase {5¢-[
32
P](pT)
8
}, ATPase ([c-
32
P]ATP), and amylase (EPS)
activities as in Fig. 1. The examples of determinations of DNase
(agarose electrophoresis), ATPase (TLC), phosphatase (TLC), RNase
(PAGE) and amylase (TLC) are given on the right (for details, see
Materials and methods). Lane numbers correspond to the numbers of
eluate fractions; C, substrate alone.
3356 T. G. Kanyshkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003
possesses two DNA-binding sites with different affinities
for oligonucleotides, which can be labeled after incubation
with affinity probes for DNA-binding and RNA-binding
sites, the 2¢,3¢-dialdehyde derivatives of different specific
and nonspecific [5¢-
32
P]oligonucleotides, including [5¢-
32
P]-
d(pT)
9
r(pU) and [5¢-
32

P](pU)
10
[24]. These modifications
fulfiled the known criteria of affinity modification [23,24].
As judged by SDS/PAGE analysis, both the LF polypeptide
purified using anti-LF–Sepharose and the combined LF-1–4
subfractions were specifically affinity-labeled by
32
Panalogs
of [5¢-
32
P]d(pT)
9
r(pU) and [5¢-
32
P](pU)
10
oligonucleotides,
and by a-[
32
P]oxATP and showed 1.4, 1.2, and 1.0 binding
sites per LF molecule, respectively (Fig. 3A). As the sample
preparation for SDS/PAGE dissociates any protein
complex, and the electrophoretic mobility of hypothetical
contaminating DNases, RNases, phosphatases, and ATP-
ases could not possibly all coincide with that of LF, the
detection of a
32
P-labeled band in the gel region
corresponding to LF, together with the absence of any

other labeled bands, provides direct evidence that LF
does not contain contaminating enzymes. In addition,
immobilized LF antibodies bound LF labeled by these
affinity reagents almost completely (data not shown).
A further approach provided direct evidence that LF
possesses five different enzyme activities. DNase and RNase
activities of the LF polypeptide were shown by in-gel in situ
assays after SDS/PAGE in gels containing DNA or RNA
(Fig. 3). Staining with ethidium bromide after development
of nuclease activity revealed a sharp dark band on a
fluorescent background of DNA or RNA (Fig. 3A, lanes
6and7).
We also used an in-gel ATPase assay, adapted from
Gomori’s method of P
i
precipitation used previously for
histochemical study of ATPase activity [32], for in situ
detection of enzyme-dependent formation of P
i
in SDS/
polyacrylamide gels after establishing conditions for preci-
pitation of Pb
2
(PO
4
)
3
in regions of gels containing P
i
and for

efficient removal of Pb salts nonspecifically adsorbed to
proteins. Enzyme-dependent formation of Pb
2
(PO
4
)
3
,detec-
ted by autoradiography, showed a
32
P-labeled product only
in the band corresponding to LF (Fig. 3A, lane 8).
Phosphatase (lane 9) and amylase (lane 10) activities of
LF were also shown by in-gel assays (Fig. 3A).
These results were obtained using a mixture of separated
subfractions LF-1 to LF-4 (Fig. 2) from the milk of three
different donors. In addition, we analyzed, using in situ
Table 1. Relative activity of different subfractions of LF obtained by chromatography on Cibacron Blue–Sepharose (Fig. 2). Thedatashowtherelative
activity of different subfractions of LF from the milk of one donor (Fig. 2) and the range of variation in the relative activities of LF subfractions
purified from milk of seven different donors (in the parentheses). In all cases, the activity of one subfraction with maximal activity was taken as
100% and the activity of other subfractions was calculated as a percentage of that with maximal activity. Zero indicates the absence of any activity
in the subfraction analyzed, but in some cases there may be detectable activity from closely positioned peaks of activity.
Enzyme activity
Relative activity of different LF fractions (%)
Additional dataLF-1 LF-2 LF-3 LF-4 LF-5
DNase 100 (100) 0 (0–5) 0 (0) 33 (22–41) 0 (0) 16 (7–20), between LF-2 and LF-3
ATPase 100 (100) 0 (0) 0 (0) 0 (0) 53 (39–62) –
Phosphatase 63 (41–68) 26 (17–36) 100 (100) 17 (8–25) 0 (0) –
RNase 100 (100) 44 (31–49) 63 (45–69) 0 (0) 0 (0) –
Amylase 0 (0) 0 (0) 37 (23–40) 0 (0–6) 100 (100) –

Fig. 3. In-gel detection of enzyme activities of the LF polypeptide, its
tryptic fragments and proteins of human milk in SDS/12% polyacryl-
amide gels. (A) Lane 1, silver stained; lane 2, immunoblot (alkaline
phosphatase-conjugated anti-LF); lanes 3–5, LF affinity-labeled by
periodate-oxidized [a-
32
P]ATP (3), 5¢-[
32
P](pU)
10
(4), or 5¢-[
32
P]-
d(pT)
9
r(pU) (5) (autoradiographs); lanes 6–7, (the negatives of the
films are shown), DNase and RNase in gels containing calf thymus
DNA (6) or yeast RNA (7); lane 8, ATPase; lane 9, phosphatase; lane
10, amylolytic activity (RA, relative activity), respectively {2–3 mm gel
slices incubated with 5¢-[
32
P](pT)
8
or EPS}. (B) Lanes 1 and 2,
Comassie R250-stained LF (1) and its tryptic fragments (2); lanes 3–6,
the negatives of the films corresponding to DNase (3, 4), RNase (5) and
ATPase {6; [
32
P]Pb
3

(PO
4
)
2
activity of LF (3) and its tryptic fragments
(4–6)}. (C) In situ analysis of DNAse (lanes 1, 2), RNase (lanes 3, 4) and
ATPase (lanes 5, 6) of human milk proteins (3–7 lL human plasma);
Comassie R250-stained proteins (1, 3, 5), DNase (2), RNase (4) (the
negatives of the films) and ATPase (6) activity {[
32
P]Pb
3
(PO
4
)
2
}.
Ó FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3357
detection of enzyme activities of separated LF-1 (DNAse,
RNase, ATPase), LF-3 (phosphatase, RNase, amylase),
and LF-5 (ATPase, amylase), subfractions corresponding to
LF from two donors, and obtained the the same result as for
the LF-1–LF-4 mixture (data not shown).
Mild treatment of LF with trypsin at pH 8.2 cleaves the
molecule between Lys283 and Ser284 into a N-tryptic lobe
(molecular mass  30 kDa) and C-tryptic (molecular mass
 50 kDa) fragment [33]. The high-affinity DNA-binding
site is located in the N-domain of LF [24], and the ATP-
binding site in the C-terminal domain [23]. We obtained
these fragments by tryptic hydrolysis of LF (not fraction-

ated on Cibacron Blue–Sepharose) and analyzed their
activities by SDS/PAGE. Figure 3B shows that the
N-tryptic fragment catalyses the hydrolysis of DNA and
RNA, whereas the C-terminal domain is responsible for the
hydrolysis of ATP. In addition, modification of LF with
oxATP did not lead to a decrease in its DNase and RNase
activities (data not shown). This result is consistent with the
localization of nucleic acid-binding and ATP- binding sites
in the N and C lobes, respectively [23,24]. Together, these
observations show that all five enzyme activities are intrinsic
properties of LF.
Substrate specificity of LF
Fractions of LF with maximal activity in each of the five
enzymatic reactions (Fig. 2) were used for more detailed
studies. LF DNase had properties that distinguished it
clearly from other known DNases. Its pH optimum was
7.0–7.5, a value markedly higher than that (5.0–5.5) [25,30]
of human blood DNase II, and the activity was significantly
(100–150%) activated by 100 m
M
NaCl whereas DNase I
is 70% inhibited by 50 m
M
NaCl [25,30]. Cleavage of
oligonucleotides and DNA by LF was stimulated 3–5-fold
by Ca
2+
,Cu
2+
,andZn

2+
and8–9-foldbyMn
2+
and
Mg
2+
ions. In contrast with known human DNases, LF
DNase was activated by ATP, dATP and NAD (150 m
M
)
by a factor of 1.5–2.5 (data not shown).
Subfraction LF-1 from the milk of different donors
cleaved the deoxyribo-oligonucleotides GGCACTTAC,
TAGAAGATCAAA, and ACTACACATCTACA, corres-
ponding to sequences to which it is known to bind
and activate transcription [10], as well as different d(pN)
10
with comparable K
m
values (3.7–7.2 l
M
)butwith
different efficiencies (k
cat
¼ 0.006–0.042 min
)1
; Table 2).
Interestingly, K
m
and k

cat
for different homo-d(pN)
10
and
homo-(pN)
10
(K
m
¼ 3.0–5.0; k
cat
¼ 0.026–0.029 min
)1
)
were comparable (Table 2). The K
m
values for different
homo-d(pN)
10
and homo-(pN)
10
molecules were also
comparable (a difference within 40%) for LF-1 subfractions
of nonfractionated LFs from milk of seven different
donors (data not shown). More significant differences were
observed for k
cat
values for LF-1 and nonfractionated
LF in milk from different donors, but these values
correlate with the variation in the relative DNase and
RNase activities of nonfractionated LF preparations and

their LF-1 subfractions and the relative amounts of LF-1
in total LF [the main LF-5 fraction (80–90%) does not
possess DNase activity].
The LF-1 fraction from milk of different donors cleaved
plasmid DNAs (phage k, pBR-322, Bluescript) 30–200
timesfaster(k
cat
¼ 2–9 min
)1
) than the oligonucleotides, a
rate comparable to that of some DNA restriction endonuc-
leases [25]. A similar result was obtained for the relative
activities of nonfractionated LF in the hydrolysis of
oligonucleotides and plasmid DNA.
RNase activity has been reported previously in human
milk LF [8,9], and we found in the present study that its
substrate specificity distinguishes it from RNase A and all
other human sera and milk RNases, as shown by its pattern
of cleavage of tRNA
Phe
. It showed major cleavage sites in
the double-stranded UGUG region between nucleotides 47
and 48, 50 and 51, and especially 52 and 53, which are
unique (Fig. 4B). The data on the difference in tRNA
hydrolysis by LF and RNase A are summarized in Fig. 4C.
Seven LF-1 preparations from different donors hydro-
lyzed ATP with K
m
¼ 0.5 ± 0.2 m
M

,andthek
cat
values
varied in the range (0.5–4) · 10
)3
min
)1
.TheK
m
(ATP)
values for LF-1 preparations do not differ significantly
from those for corresponding nonfractionated LFs (K
m
¼
0.2–1.0 m
M
).
Of the nine oligosaccharides studied, only malto-oligo-
saccharide was hydrolyzed by different nonfractionated LF
preparations. The LF-5 fraction from one milk donor
hydrolyzed malto-oligosaccharide with a K
m
¼ 2.0 m
M
and
specific activity of 10 ± 2 standard units/mg. The K
m
values (2.0 ± 0.9 m
M
) for malto-oligosaccharide in the case

of seven different LF-5 subfractions and the seven corres-
ponding nonfractionated LF preparations were compar-
able, their specific activities depending slightly on the milk
donor and varying in the range 5–17 standard units per mg.
These results agree with the fact that subfraction LF-5
(peak 4, Fig. 2) constitutes  80–90% of total LF.
Table 2. K
m
and k
cat
values for different ribo-oligonucleotide and deoxyribooligonucleotides characterizing their hydrolysis by LF-1 subfractions of LF
from seven samples of different human milk. Results are mean ± SD from three measurements for each of seven LF preparations.
Substrate K
m
(l
M
)10
3
· k
cat
(min
)1
)10
)3
· k
cat
/K
m
(min
)1

Æ
M
)1
)
d(pT)
10
5.6 ± 2.0 42.8 7.6
d(pA)
10
4.2 ± 1.5 6.4 ± 2.0 1.5
d(pN
˜
)
10
4.5 ± 1.3 28.1 ± 7.0 6.3
(pU)
10
3.4 ± 1.0 29.6 ± 6.3 8.8
(pA)
10
3.0 ± 1.0 26.0 ± 7.9 8.6
(pC)
10
5.0 ± 2.1 29.6 ± 9.1 5.9
d(pGpGpCpApCpTpTpApC) 7.2 ± 1.1 27.2 ± 5.7 3.8
d(pTpApGpApApGpApTpCpApApA) 3.7 ± 1.0 8.2 ± 2.5 2.2
d(pApCpTpApCpApGpTpCpTpApCpA) 5.3 ± 1.8 42.2 ± 4.1 8.0
3358 T. G. Kanyshkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003
LF as the major DNAse, RNase, and ATPase
of human milk

The hydrolytic function of an enzyme can have a protective
role in prokaryotic and eukaryotic cells, and we therefore
compared the activities of LF in the hydrolysis of DNA,
RNA, and ATP with those of other DNases, RNases, and
ATPases of human milk. Published data on these activities
in human milk are limited. The14-kDa RNase so far
reported [8], like five human blood 14–25-kDa RNases with
different affinity for phosphocellulose [37], is relatively small
and has substrate specificity similar to that of pancreatic
14-kDa RNase A [38]. Only one 42-kDa DNase with
catalytic properties similar to that of DNase II has been
described in human blood [39]. In addition, we have recently
presented evidence that the milk of healthy human mothers
contains subfractions of 150-kDa IgG and 360-kDa sIgA
antibodies, which hydrolyze DNA, RNA, and ATP [25,35].
After separation of human milk proteins by SDS/PAGE
in a gel containing DNA, an in-gel assay showed DNase
activity mostly in the protein band corresponding to LF
(Fig. 3C, lane 4). In contrast, DNase activity corresponding
to human milk 41-kDa DNAse II (Fig. 3C, lane 4) was
detected only in half of 14 analyzed milk samples. A similar
situation was observed after separation of human milk
proteins in a gel containing RNA; again LF was signifi-
cantly more active in hydrolysing RNA than 14-kDa RNase
or antibodies (Fig. 3C, lane 2). LF was also significantly
more active in hydrolysing ATP than IgG and sIgA
antibodies. Furthermore, under the conditions used, we
did not detect any other ATPases or phosphatases of low
molecular mass (Fig. 3C, lane 6). Thus, LF is the predomi-
nant DNase, RNase, and ATPase in human milk, and it is

likely that LF may have a protective function during breast
feeding of the newborn as well as in human epithelial
secretions and barrier fluids during viral and bacterial
infections.
Cytotoxicity and apoptosis-inducing activities of LF
For breast-fed infants, human milk is more than a source of
nutrients; it furnishes a wide array of antimicrobial and
antiviral molecules, and may also contain substances
bioactive toward host cells; for example, it is cytotoxic to
human cancer cells [40] because of induction of apoptosis by
multimeric a-lactalbumin.
We studied the effects of various subfractions of LF with
catalytic activities on the growth of L929 (mouse fibroblasts)
and HL-60 (human promyelocytes) cells. No effect of the
main LF-5 fraction, which possesses only ATPase and
amylase activities, was detected [5 mgÆmL
)1
; corresponding
to peak 4 (LF-5); Fig. 2], but fraction LF-1 [peak 2 (LF-1);
Fig. 2], which possesses at least four enzymic activities
(DNase, RNase, phosphatase, and ATPase), showed high
cytotoxicity, only 1.5–1.7 times lower than that of tumor
necrosis factor (Fig. 5A). All other catalytically active
subfractions of LF-2–4 (peaks 3–4) were also cytotoxic,
but their effects were estimated as  10–50% of that of
LF-1. As LF-5 has ATPase and malto-oligosaccharide-
hydrolyzing activities but is not cytotoxic, the cytotoxicity of
LF-1 to LF-4 is probably associated with their DNase and/
or RNase activities.
DNA of tumor cell lines L929 and HL-60 exposed to

fraction LF-1 (peak 2, Fig. 2) for 12–24 h was fragmented
as the concentration of the LF increased and became
significant at 100 n
M
(Fig. 5B), and oligonucleosome-size
DNAs fragments typical of apoptosis [41,42] were formed.
Cells exposed to subfraction LF-1 also display annexin
V-staining and morphological changes typical of apoptosis
(Fig. 5C).
Discussion
Here we show for the first time that subfractions obtained
by chromatography of homogeneous human milk LF on
Cibacron Blue–Sepharose possess different catalytic acti-
vities and that DNase, RNase, phosphatase, ATPase, and
amylase activities are intrinsic properties of human milk LF.
Chromatographic separation (Fig. 2) indicates that these
five activities are associated with different isoforms of LF
and that, in addition to the three previously reported
isoforms [9], one of which is LF RNase, further isoforms of
this protein exist that possess other enzyme activities. The
nature of the structural variations that give rise to these
Fig.4.EnzymeactivitiesofLFrecoveredbygelfiltrationonaTSK
HW-55 column. (A) Enzymatic activities of LF recovered by gel fil-
tration on a TSK HW-55 column in Tris/glycine (pH 2.4)/0.3
M
NaCl
after incubation for 30 min at 25 °C in the same buffer to dissociate
noncovalently bound proteins; solid line, A
280
; symbols, relative activity

assayed as in Fig. 1. (B) Partial cleavage of human 3¢-[
32
P]tRNA
Phe
by
LF (lane 2) compared with cleavage by RNase A (lane 1); lane 3, tRNA
incubated alone. (C) The cloverleaf structure of tRNA
Phe
showing the
major cleavage sites for LF and RNase A. Symbol size (and intensity)
corresponds to the relative hydrolytic activity.
Ó FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3359
profound functional differences is not known. The LF
molecule contains two potential glycosylation sites [2], the
degree of glycosylation of different molecules varies, and
they can contain hexose, mannose, hexosamines, or other
saccharides [43] and may also differ in the level of
phosphorylation.
According to X-ray crystallographic analysis, LF consists
of two lobes joined by a very flexible amino-acid spacer [1]
and is therefore extremely conformationally flexible; its
functional state can be influenced not only by iron ions [44]
but also by other metal ions and different ligands such as
DNA, RNA, and polyanions [45]. Specific conformations of
monomeric LF induced by different ligands may modulate
its enzymatic functions; binding of DNA is not a rapid
process but requires preincubation, and ligands such as
ATP and NAD significantly influence this process [45]. The
nature of the intersubunit interactions in LF oligomers
remains unknown and most investigators do not take into

consideration its oligomeric forms. Considering the relat-
ively small size of the monomer and its polyfunctionality,
we suggested that it may have various oligomeric forms
(monomer, dimer, trimer, and tetramer) [23], the intercon-
version of which may be controlled by ATP and/or other
low-molecular-mass ligands. ATP binding modifies
oligomerization which is accompanied by changes in
interaction with nucleic acids, polysaccharides, and proteins.
Furthermore, as shown above, ATP and NAD stimulate
LF-dependent hydrolysis of DNA. Thus, ATP-dependent
changes in the oligomeric structure may increase the number
of functional states and biological functions of LF. Further
study of the structure of its catalytic isoforms and its
relation to their various functions is required.
Our data indicate that the subfractions of LF constitute
the major DNase, RNase, and ATPase in human milk
(Fig. 3C) and therefore may contribute to its protective
functions; injection of nucleases into the circulatory system
or treatment of human respiratory mucosal surfaces with
DNases and RNases leads to protection against viral and
bacterial diseases [46]. Recently, an inverse correlation
between mammary tumor incidence and the amount of
RNase activity in human milk was revealed [8]. Several
catalytically active LF subfractions are cytotoxic (Fig. 5A),
and the most active, the effect of which is comparable to
that of tumor necrosis factor, is LF-1, which possesses the
highest DNAse, RNase, and ATPase activities. This frac-
tion was capable of inducing apoptosis, raising the pos-
sibility that LF contributes to mucosal immunity not only
by its antimicrobial and antiviral properties but also by

policing the function of human cells. The discovery of five
enzymatically distinct forms of LF, their cytotoxicity, and
the ability of some of them to induce tumor cell apoptosis
should contribute to the understanding of its remarkable
polyfunctional activities.
Acknowledgements
This research was made possible in part by a grant from RFBR (98-04-
49719 and 01-04-49759) and a grant for young scientists from the
Siberian Branch of RAS.
References
1. Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W. & Baker,
E.N. (1989) Structure of human lactoferrin: crystallographic
structure analysis and refinement at 2.8 A
˚
resolution. J. Mol. Biol.
209, 711–734.
2. Van Berkel, P.H., van Veen, H.A., Geerts, M.E., de Boer, H.A. &
Nuijens, J.H. (1996) Heterogeneity in utilization of N-glycosyla-
tion sites Asn624 and Asn138 in human lactoferrin: a study with
glycosylation-site mutants. Biochem. J. 319, 117–122.
3. Masson, P.L., Heremans, J.F. & Schonne, E. (1969) Lactoferrin,
an iron-binding protein in neutrophilic leukocytes. J. Exp. Med.
130, 643–453.
4. Brock, J.H. (1985) Transferrins. In Topics in Molecular and
Structural Biology: Metalloproteins (Harrison, P.M., ed.) Chemie
Verlag, Weinheim.
5. De Sousa, M. & Brock, J.H. (1989) Iron in Immunity Cancer and
Inflammation. John Wiley and Sons, New York.
6. Bennett, R.M., Merrit, M.M. & Gabor, G. (1986) Lactoferrin
binds to neutrophilic membrane DNA. Br.J.Haematol.63,

105–117.
7. Zimecki, M., Mazurier, J., Spik, G. & Kapp, J.A. (1995) Human
lactoferrin induces phenotypic and functional changes in murine
splenic B cells. Immunology 86, 122–127.
8. Ramaswamy, H., Swamy, C.V. & Das, M.R. (1993) Purification
and characterization of a high molecular weight ribonuclease from
human milk. J. Biol. Chem. 268, 4181–4187.
9. Furmanski, P., Li, Z.P., Fortuna, M.B., Swamy, C.V. & Das,
M.R. (1989) Multiple molecular forms of human lactoferrin.
Fig. 5. Cytoxicity of LF. (A) Loss of viability (trypan blue staining) of
L929 cells during incubation with 10 n
M
tumor necrosis factor (curve
1), 100 n
M
LF- 1 (curve 2) or LF-4 fraction (curve 3); curve 4, control.
(B) Chromatin cleavage induced by LF in L929 cells. The cells were
either untreated (lane 1) or treated with 5 mgÆmL
)1
LF-5 (lane 2) or
100 n
M
LF-1 from three different milk donors (lanes 3–5) for 18 h.
DNA was electrophoresed in 1.2% agarose gel and stained with
ethidium bromide. Lanes 1 and 2 show no detectable fragmentation,
whereasLF-1(lanes3–5)causedaÔladderÕ of DNA fragments. Posi-
tions of size standards are shown on the right. (C) Apoptosis of HL-60
human cells treated with 100 n
M
LF-1 for 16 h revealed by annexin V

staining.
3360 T. G. Kanyshkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Identification of a class of lactoferrins that possess ribonuclease
activity and lack iron-binding capacity. J. Exp. Med. 170, 415–429.
10. He, J. & Furmanski, P. (1995) Sequence specificity and tran-
scriptional activation in the binding of lactoferrin to DNA. Nature
(London) 373, 721–724.
11. Fleet, J.C. (1995) A new role for lactoferrin: DNA binding and
transcription activation. Nutr. Rev. 53, 226–227.
12. Mantel, C., Miyazawa, K. & Broxmeyer, H.E. (1994) Physical
characteristics and polymerization during iron saturation of
lactoferrin, a myelopoietic regulatory molecule with suppressor
activity. Adv. Exp. Med. Biol. 357, 121–132.
13. Bezault, J., Bhimani, R., Wiprovnick, J. & Furmanski, P. (1994)
Human lactoferrin inhibits growth of solid tumors and develop-
ment of experimental metastases in mice. Cancer Res. 54, 2310–
2312.
14. Sawatzki, G. & Rich, I.N. (1989) Lactoferrin stimulates colony
stimulating factor production in vitro and in vivo. Blood Cells 15,
371–385.
15. Gahr, M., Speer, C.P., Damerau, B. & Sawatzki, G. (1991)
Influence of lactoferrin on the function of human polymorpho-
nuclear leukocytes and monocytes. J. Leukocyte Biol. 49, 427–433.
16. De Sousa, M., Breedvelt, F., Dynesius-Trentham, R., Trentham,
D. & Lum, J. (1988) Iron, iron-binding proteins and immune
system cells. Ann. NY Acad. Sci. 526, 310–322.
17. Amouric,M.,Marvaldi,J.,Pichon,J.,Bellot,F.&Figarella,C.
(1984) Effect of lactoferrin on the growth of a human colon
adenocarcinoma cell line-comparison with transferrin. In Vitro 20,
543–548.

18. Garre, C., Bianchi-Scarra, G., Sirito, M., Musso, M. &
Ravazzolo, R. (1992) Lactoferrin binding sites and nuclear loca-
lization in K562 (S) cells. J. Cell Physiol. 153, 477–482.
19. Arnold, R.R., Cole, M.F. & McGhee, F.M. (1977) A bactericidal
effect for human lactoferrin. Science 197, 263–265.
20. Yi,M.,Kaneko,S.,Yu,D.V.&Marakami,S.(1977)HepatitisC
virus envelope proteins bind lactoferrin. J. Virol. 71, 5997–6002.
21. Ellison, R.T., Giehl, T.J. & Laforse, F.M. (1988) Damage of the
outer membrane of enteric gram-negative bacteria by lactoferrin
and transferring. Infect. Immun. 56, 2774–2780.
22. Semenov, D.V., Kanyshkova, T.G., Buneva, V.N. & Nevinsky,
G.A. (1998) Interaction of human milk lactoferrin with ATP.
Biochemistry (Mosc) 63, 67–75.
23. Semenov, D.V., Kanyshkova, T.G., Buneva, V.N. & Nevinsky,
G.A. (1999) Human milk lactoferrin binds ATP and dissociates
into monomers. Biochem. Mol. Biol. Int. 47, 177–184.
24. Kanyshkova, T.G., Semenov, D.V., Buneva, V.N. & Nevinsky,
G.A.(1999)HumanmilklactoferrinbindstwoDNAmolecules
with different affinities. FEBS Lett. 451, 235–237.
25. Nevinsky, G.A., Kanyshkova, T.G., Semenov, D.V., Vlassov,
A.V., Gal’vita, A.V. & Buneva, V.N. (2000) Secretory immuno-
globulin A from healthy human mothers’ milk catalyzes nucleic
acid hydrolysis. Appl. Biochem. Biotechnol. 83, 115–130.
26. Perret, V., Garcia, A., Puglisi, J.D., Grosjean, H. & Ebel, J P.
(1990) Conformation in solution of yeast tRNA (Asp) transcripts
deprived of modified nucleotides. Biochimie 72, 735–743.
27.Vlassov,V.V.,Zuber,G.,Felden,B.,Behr,J P.&Giege,R.
(1995) Cleavage of tRNA with imidazole and spermine imidazole
constructs: a new approach for probing RNA structure. Nucleic
Acids Res. 23, 3161–3167.

28. Savel’ev, E.V., Eneyskaya, K.A., Shabalin, M.V., Filatov, M.V. &
Neustroev, K.N. (1999) Antibodies with amylolytic activity.
Protein Peptide Lett. 6, 179–184.
29. Kanyshkova, T.G., Semenov, D.V., Khlimankov, Yu, D.,
Buneva, V.N. & Nevinsky, G.A. (1997) DNA-hydrolyzing activity
of the light chain of IgG antibodies from milk of healthy human
mothers. FEBS Lett. 416, 23–26.
30. Buneva, V.N., Kanyshkova, T.G., Vlassov, A.V., Semenov,
D.V., Khlimankov, D.Y., Breusova, L.R. & Nevinsky, G.A.
(1998) Catalytic DNA- and RNA-hydrolyzing antibodies from
milk of healthy human mothers. Appl. Biochem. Biotechnol. 75,
63–76.
31. Andrievskaia, O.A., Buneva, V.N., Naumov, V.A. & Nevinsky,
G.A. (2000) Catalytic heterogenity of polyclonal RNA-hydrolyz-
ing IgM from sera of patients with lupus erythematosus. Med. Sci.
Monit. 6, 460–470.
32. Burstone, M.S. (1962) Enzyme Histochemistry and its Application
in the Study of Neoplasms.AcademicPress,NewYorkand
London.
33. Legrand, D., Mazurier, J., Metz-Boutigue, M., Jolles, J., Jolles, P.,
Montreuil, J. & Spik, J. (1984) Characterization and localization
of an iron-binding 18-kDa glycopeptide isolated from the
N-terminal half of human lactotransferrin. Biochim. Biophys. Acta
787, 90–96.
40. Hakasson, A., Zhivotovsky, B., Orrenius, A., Sabharwal, H. &
Svanborg, C. (1995) Apoptosis induced by a human milk protein.
Proc.Nat.Acad.Sci.USA92, 8064–8068.
34. Zhivotovsky, B., Wade, D., Gahm, A., Orrenius, S. & Nicotera, P.
(1994) Formation of 50 kbp chromatin fragments in isolated liver
nuclei is mediated by protease and endonuclease activation. FEBS

Lett. 351, 150–154.
35. Vlassov, A., Florentz, C., Helm, M., Naumov, V., Buneva, V.,
Nevinsky, G. & Giege, R. (1998) Characterization and selectivity
of catalytic antibodies from human serum with RNase activity.
Nucleic Acids Res. 26, 5243–5250.
36. Nevinsky,G.A.,Kit,Yu,Ya.,Semenov,D.V.&Buneva,V.N.
(1998) Secretory immunoglobulin A from human milk catalyzes
milk protein phosphorylation. Appl. Biochem. Biotechnol. 75,
77–91.
37. Akagi, K., Murai, K., Hirao, N. & Yamanaka, M. (1976) Puri-
fication and properties of alkaline ribonuclease from human
serum. Biochim. Biophys. Acta 442, 368–378.
38. De, I., Cardayre, S.B. & Raines, R.T. (1995) A residue to residue
hydrogen bond mediates the nucleotide specificity of ribo-
nuclease A. J. Mol. Biol. 252, 328–336.
39. Gudding, R. (1979) DNases in milk and blood sera from different
species. Acta Vet. Scand. 20, 404–416.
41. Schwartz, L.M. & Osborne, B.A. (1993) Programmed cell death,
apoptosis and killer genes. Immunol. Today 14, 582–590.
42. Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis
is associated with endogenous endonuclease activation. Nature
(London) 284, 555–556.
43. Legrand, D., Salmon, V., Coddeville, B., Benaissa, M., Plancke,
Y. & Spik, G. (1995) Structural determination of two N-linked
glycans isolated from recombinant human lactoferrin expressed in
BHK cells. FEBS Lett. 365, 57–60.
44. Anderson, B.F., Baker, H.M., Norris, G.E., Rumball, S.V. &
Baker, E.N. (1990) Apolactoferrin structure demonstrates ligand-
induced conformational change in transferrins. Nature (London)
344, 784–787.

45. Kanyshkova, T.G., Buneva, V.N. & Nevinsky, G.A. (2001)
Lactoferrin and its biological functions. Biochemistry (Mosc) 66,
1–7.
46. Nevinsky, G.A., Favorova, O.O. & Buneva, V.N. (2002) Natural
catalytic antibodies: new characters in the protein repertoire.
In Protein–Protein Interactions: A Molecular Cloning Manual
(Golemis, E., ed.), pp. 523–534. Cold Spring Harbor Laboratory
Press, New York.
Ó FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3361