Lactoferrin (LF) is a single polypeptide chain of 76–80 kDa, cell and nuclear membrane [18]. Owing to its antiviral and
containing two lobes [1], each of which binds one Fe3+ ion antimicrobial activities, LF increases the passive immunity
and contains one glycan chain [2]. It was first recognized of newborns. It was initially suggested that the antimicro-
in milk and then in other human epithelial secretions and bial properties of LF may be attributed to its iron-binding
barrier body fluids [3–6]. Many different functions have capacity; removal of iron from the microbial environment
been attributed to LF, including protection from iron- is an important defense mechanism as it is needed for the
induced lipid peroxidation, immunomodulation and cell proliferation of microflora [19]. Many micro-organisms
growth regulation [6,7], DNA binding [6], RNA hydrolysis express surface receptors for LF and it may show different
[8,9], and transcriptional activation of specific DNA iron-independent antimicrobial and antiviral properties
sequences [10,11]. It is a potent activator of natural killer [20,21], the mechanisms of which are still a matter of
cells [12] and may have an antitumor role [7,13], an activity debate.
that is independent of iron. LF also influences granulo- We have proposed that, as LF is a relatively small
poiesis [14], antibody-dependent cytotoxicity [15], cytokine protein, its polyfunctional properties may result from its
production [16], and growth of some cells in vitro [17]. The existence in several oligomeric forms that have different
physiological role of LF and the mechanisms underlying activities, and that its oligomerization and dissociation are
these activities are still unclear, but it has been suggested to under the control of specific ligands such as ATP [22,23].
be responsible for primary defense against microbial and In support of this idea, we have shown recently that LF
viral infection [3,5]. LF is a protein of the acute phase; the possesses an ATP-binding site and that interaction of the
highest concentration is usually detected in the inflamma- protein with ATP leads to changes in its interaction with
tory nidus. It is detected in the blood of newborn babies polysaccharides, DNA and proteins [23]. We have further
several hours after feeding, and can readily penetrate any 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
Correspondence to G. A. Nevinsky, Laboratory of Repair Enzymes, the known polyanion-binding and antimicrobial domains of
Novosibirsk Institute of Bioorganic Chemistry, 8, Lavrentieva Ave., the protein [24].
630090, Novosibirsk, Russia. Here we show that this extremely polyfunctional protein
Fax: 007 3832 333677, Tel.: 007 3832 396226, possesses five enzyme activities (DNase, RNase, ATPase,
E-mail: nevinsky@niboch.nsc.ru phosphatase, and malto-oligosaccharide hydrolysis). The
Abbreviations: LF, human milk lactoferrin; EPS, 4-nitrophenyl RNA-hydrolyzing and DNA-hydrolyzing subfractions of
4,6-O-ethylidene-a-D-maltoheptaoside. LF may contribute to its protective role through hydrolysis
(Received 23 January 2003, revised 13 May 2003, of viral and bacterial nucleic acids. In addition, we show
accepted 11 June 2003) that some catalytic forms of LF are cytotoxic and
3354 T. G. Kanyshkova et al. (Eur. J. Biochem. 270) FEBS 2003
apoptosis-inducing agents. These findings suggest that LF Phosphatase activity was assayed under the same condi-
of milk and other human epithelial secretions and body tions: removal of [32P]Pi from 5¢-[32P]oligonucleotides was
fluids may contribute to cell defense by policing the function assayed by TLC in dioxane/NH4OH/water (5 : 1 : 4, by
of human cells. vol.) on Kieselgel plates (Merck). After chromatography,
the plates were dried, the [32P]products localized by
autoradiography, and their radioactivity was measured by
Materials and methods Cherenkov counting.
The same conditions were used to study cleavage of
Materials and chemicals
human tRNAPhe prepared as described previously [26,27]
Reagents were obtained mainly from Sigma and Merck. and labeled at the 5¢ end [26,27]. tRNA (0.1 lgÆmL)1; 105
4-Nitrophenyl 4,6-O-ethylidene-a-D-maltoheptaoside (EPS) Cherenkov counts per sample) were incubated at 37 C for
was purchased from Boehringer Mannheim (Germany). 30 min with LF (0.1–1.0 lM) or RNase A (5 · 10)5
We also used heparin, antibodies to human LF (Sigma), mgÆmL)1), and the products were analyzed by electropho-
DEAE-cellulose DE-52 (Whatman), heparin–Sepharose resis in 15% polyacrylamide/8 M urea gels, with partial
and Cibacron Blue–Sepharose CL-6b (Pharmacia Fine RNase T1 and imidazole digests of the tRNAs run in
Chemicals), and Toyopearl HW-55 fine (Toyo Soda). parallel to identify the products [27]. Quantification was
Radioisotopes were purchased from Amersham performed by analysis on a Fujix BioImaging Analyzer
(3000 CiÆmmol)1). BAS 2000 System (Fuji).
washed with a solution of 4 M urea and twice with water to and visualized with ethidium bromide [34]. An Annexin-V-
remove SDS, and then to allow protein renaturation it was Fluorescein kit was used for analysis of apoptosis according
incubated for 16 h at 37 C in 20 mM Tris/HCl buffer, to instructions provided by the manufacturer (Boehringer-
pH 7.5, containing 1 mM EDTA and 5.0 mM MgCl2. To Mannheim).
reveal the regions of DNA or RNA hydrolysis, the gel was
stained with ethidium bromide. Proteins were revealed by
Coomassie R250 staining.
Results
ATPase was detected using our modification of the
Purification and characterization of LF subfractions
Gomori method for histochemical determination of ATP-
ases [32]. After electrophoresis, SDS was removed by We isolated and analyzed separately LF preparations from
incubating the gel for 30 min at 37 C with water (5 times) the milk of 30 different healthy mothers. LF was purified
and then with 0.5 M sodium acetate, pH 6.8 (3 times). To from the fraction of human milk that was not adsorbed by
allow protein renaturation and to detect Pi resulting from DEAE-cellulose by chromatography on heparin–Sepharose
ATP hydrolysis, the gel was incubated for 12 h at 37 C in [22–24], and electrophoretically homogeneous LF was
5 mM sodium acetate (pH 6.8) containing 1.0 mM MgCl2, purified on anti-LF–Sepharose (Fig. 1). As shown previ-
3 mM Pb(NO3)2, and 100 lCi [c-32P]ATP. Nonspecifically ously [9], human milk LF could be separated into several
adsorbed Pb(NO3)2 was removed by washing the gel 3 times distinct isoforms by affinity chromatography on Cibacron
(10 min) with water, then with hot 5% acetic acid and again Blue–Sepharose. We found that chromatographically,
with water. The gel was autoradiographed to detect [32P]Pi. electrophoretically, and immunologically homogeneous
Phosphatase and amylase activity was determined using LF (after anti-LF–Sepharose chromatography) contains
gels without substrates. After electrophoresis, SDS was subfractions with different affinities for Cibacron Blue–
removed by incubating the gels as for analysis of DNase Sepharose (Fig. 2A–C). They all possessed the N-terminal
activity. The gels were then cut into 2 mm slices which were amino-acid sequence reported for LF, Gly-Arg-Arg-Arg-
incubated with 20 mM Tris/HCl, pH 7.5, at 4 C for 12 h. Arg-Ser-Val-Glu [9], and also a product of partial proteo-
The gel slices were removed by centrifugation, and amylase lytic cleavage [23,24].
or phosphatase activity was assayed using 5¢-[32P](pT)8 or Four prominent protein peaks corresponding to LF were
EPS as described above. eluted from Cibacron Blue–Sepharose (Fig. 2). The main
In situ gel assays of the enzymic activities of human milk subfraction of LF (peak 4, Fig. 2) had the highest affinity
proteins (3–7 lL dialyzed human plasma) and the limited for this sorbent. Three additional subfractions (peaks 1–3,
proteolytic cleavage products of LF (2–7 lg) were as Fig. 2) represented 10–20% of the total LF dependent on
described above for purified LF. Partial proteolytic cleavage the milk donor. The first protein peak showed no enzyme
of LF was performed using 0.1–0.5% trypsin (w/w of LF) in activity, but the three other peaks showed oligonucleotide
0.1 M Tris/HCl (pH 8.2)/25 mM CaCl2 at 37 C for 4 h [33]. 5¢-phosphatase, DNase, RNase, ATPase, and malto-oligo-
Km and Vmax for the hydrolysis of different substrates saccharide-hydrolyzing activities, each activity being eluted
were determined by the method of initial rates using in several peaks (Fig. 2A–C). The LF subfraction corres-
nonlinear regression analysis. Errors in the values were ponding to peak 2 possessed four different activities:
within 10–30%. phosphatase, DNase, RNase, ATPase. Eluate correspond-
ing to protein peak 3 showed three prominent peaks of
oligonucleotide 5¢-phosphatase activity (Fig. 2B) and two
Cytotoxicity assays
peaks of RNase activity (Fig. 2C). Interestingly, two
Tumor cell lines L929 (mouse fibroblasts) and HL-60
(human promyelocytes) were cultured at 37 C in 0.1 mL
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 nM) of subfractions of LF-1 to LF-5 (see below)
or tumor necrosis factor (10 nM) 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.
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
Fig. 2. Chromatography of LF on Cibacron Blue–Sepharose. (A) filtration. A single peak corresponding to LF was recovered
DNAse (d) and ATPase (s); (B) 5¢-oligonucleotide phosphatase (n); (see Fig. 4A), which contained 80–95% of all five enzyme
(C) RNase (*) and amylase (j). Aliquots (1–3 lL) of column fractions activities loaded on the column. There were no other peaks
were used to determine DNAse (k DNA), RNase {5¢-[32P](pU)10}, of activity or protein. The same result was obtained for
phosphatase {5¢-[32P](pT)8}, ATPase ([c-32P]ATP), and amylase (EPS) separated subfractions LF-1, LF-3 and LF-5 (Fig. 2),
activities as in Fig. 1. The examples of determinations of DNase corresponding to LF from the milk of three different
(agarose electrophoresis), ATPase (TLC), phosphatase (TLC), RNase donors (data not shown).
(PAGE) and amylase (TLC) are given on the right (for details, see Affinity labeling of enzymes with 32P analogs of their
Materials and methods). Lane numbers correspond to the numbers of
specific ligands is the most sensitive method for revealing
eluate fractions; C, substrate alone.
any contaminating proteins interacting with the same
ligands. As we showed previously, LF possesses an ATP-
additional DNase peaks were revealed in fractions 15–30 binding site, which became labeled after incubation with an
(position of peak 3), but profiles of these activity peaks did affinity probe for ATP-binding sites, the 2¢,3¢-dialdehyde
not correlate with that for protein peak 3, with the second derivative of ATP (oxATP), with a stoichiometry of 1.0 mol
and third DNase peaks occurring between protein peaks 2 [a-32P]oxATP bound per mol LF [23]. In addition, LF
FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3357
Table 1. Relative activity of different subfractions of LF obtained by chromatography on Cibacron Blue–Sepharose (Fig. 2). The data show the relative
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.
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) –
detection of enzyme activities of separated LF-1 (DNAse, homo-(pN)10 (Km ¼ 3.0–5.0; kcat ¼ 0.026–0.029 min)1)
RNase, ATPase), LF-3 (phosphatase, RNase, amylase), were comparable (Table 2). The Km values for different
and LF-5 (ATPase, amylase), subfractions corresponding to homo-d(pN)10 and homo-(pN)10 molecules were also
LF from two donors, and obtained the the same result as for comparable (a difference within 40%) for LF-1 subfractions
the LF-1–LF-4 mixture (data not shown). of nonfractionated LFs from milk of seven different
Mild treatment of LF with trypsin at pH 8.2 cleaves the donors (data not shown). More significant differences were
molecule between Lys283 and Ser284 into a N-tryptic lobe observed for kcat values for LF-1 and nonfractionated
(molecular mass 30 kDa) and C-tryptic (molecular mass LF in milk from different donors, but these values
50 kDa) fragment [33]. The high-affinity DNA-binding correlate with the variation in the relative DNase and
site is located in the N-domain of LF [24], and the ATP- RNase activities of nonfractionated LF preparations and
binding site in the C-terminal domain [23]. We obtained their LF-1 subfractions and the relative amounts of LF-1
these fragments by tryptic hydrolysis of LF (not fraction- in total LF [the main LF-5 fraction (80–90%) does not
ated on Cibacron Blue–Sepharose) and analyzed their possess DNase activity].
activities by SDS/PAGE. Figure 3B shows that the The LF-1 fraction from milk of different donors cleaved
N-tryptic fragment catalyses the hydrolysis of DNA and plasmid DNAs (phage k, pBR-322, Bluescript) 30–200
RNA, whereas the C-terminal domain is responsible for the times faster (kcat ¼ 2–9 min)1) than the oligonucleotides, a
hydrolysis of ATP. In addition, modification of LF with rate comparable to that of some DNA restriction endonuc-
oxATP did not lead to a decrease in its DNase and RNase leases [25]. A similar result was obtained for the relative
activities (data not shown). This result is consistent with the activities of nonfractionated LF in the hydrolysis of
localization of nucleic acid-binding and ATP- binding sites oligonucleotides and plasmid DNA.
in the N and C lobes, respectively [23,24]. Together, these RNase activity has been reported previously in human
observations show that all five enzyme activities are intrinsic milk LF [8,9], and we found in the present study that its
properties of LF. substrate specificity distinguishes it from RNase A and all
other human sera and milk RNases, as shown by its pattern
of cleavage of tRNAPhe. It showed major cleavage sites in
Substrate specificity of LF
the double-stranded UGUG region between nucleotides 47
Fractions of LF with maximal activity in each of the five and 48, 50 and 51, and especially 52 and 53, which are
enzymatic reactions (Fig. 2) were used for more detailed unique (Fig. 4B). The data on the difference in tRNA
studies. LF DNase had properties that distinguished it hydrolysis by LF and RNase A are summarized in Fig. 4C.
clearly from other known DNases. Its pH optimum was Seven LF-1 preparations from different donors hydro-
7.0–7.5, a value markedly higher than that (5.0–5.5) [25,30] lyzed ATP with Km ¼ 0.5 ± 0.2 mM, and the kcat values
of human blood DNase II, and the activity was significantly varied in the range (0.5–4) · 10)3 min)1. The Km(ATP)
(100–150%) activated by 100 mM NaCl whereas DNase I values for LF-1 preparations do not differ significantly
is 70% inhibited by 50 mM NaCl [25,30]. Cleavage of from those for corresponding nonfractionated LFs (Km ¼
oligonucleotides and DNA by LF was stimulated 3–5-fold 0.2–1.0 mM).
by Ca2+, Cu2+, and Zn2+ and 8–9-fold by Mn2+ and Of the nine oligosaccharides studied, only malto-oligo-
Mg2+ ions. In contrast with known human DNases, LF saccharide was hydrolyzed by different nonfractionated LF
DNase was activated by ATP, dATP and NAD (150 mM) preparations. The LF-5 fraction from one milk donor
by a factor of 1.5–2.5 (data not shown). hydrolyzed malto-oligosaccharide with a Km ¼ 2.0 mM and
Subfraction LF-1 from the milk of different donors specific activity of 10 ± 2 standard units/mg. The Km
cleaved the deoxyribo-oligonucleotides GGCACTTAC, values (2.0 ± 0.9 mM) for malto-oligosaccharide in the case
TAGAAGATCAAA, and ACTACACATCTACA, corres- of seven different LF-5 subfractions and the seven corres-
ponding to sequences to which it is known to bind ponding nonfractionated LF preparations were compar-
and activate transcription [10], as well as different d(pN)10 able, their specific activities depending slightly on the milk
with comparable Km values (3.7–7.2 lM) but with donor and varying in the range 5–17 standard units per mg.
different efficiencies (kcat ¼ 0.006–0.042 min)1; Table 2). These results agree with the fact that subfraction LF-5
Interestingly, Km and kcat for different homo-d(pN)10 and (peak 4, Fig. 2) constitutes 80–90% of total LF.
Table 2. Km and kcat 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.
Identification of a class of lactoferrins that possess ribonuclease of the light chain of IgG antibodies from milk of healthy human
activity and lack iron-binding capacity. J. Exp. Med. 170, 415–429. mothers. FEBS Lett. 416, 23–26.
10. He, J. & Furmanski, P. (1995) Sequence specificity and tran- 30. Buneva, V.N., Kanyshkova, T.G., Vlassov, A.V., Semenov,
scriptional activation in the binding of lactoferrin to DNA. Nature D.V., Khlimankov, D.Y., Breusova, L.R. & Nevinsky, G.A.
(London) 373, 721–724. (1998) Catalytic DNA- and RNA-hydrolyzing antibodies from
11. Fleet, J.C. (1995) A new role for lactoferrin: DNA binding and milk of healthy human mothers. Appl. Biochem. Biotechnol. 75,
transcription activation. Nutr. Rev. 53, 226–227. 63–76.
12. Mantel, C., Miyazawa, K. & Broxmeyer, H.E. (1994) Physical 31. Andrievskaia, O.A., Buneva, V.N., Naumov, V.A. & Nevinsky,
characteristics and polymerization during iron saturation of G.A. (2000) Catalytic heterogenity of polyclonal RNA-hydrolyz-
lactoferrin, a myelopoietic regulatory molecule with suppressor ing IgM from sera of patients with lupus erythematosus. Med. Sci.
activity. Adv. Exp. Med. Biol. 357, 121–132. Monit. 6, 460–470.
13. Bezault, J., Bhimani, R., Wiprovnick, J. & Furmanski, P. (1994) 32. Burstone, M.S. (1962) Enzyme Histochemistry and its Application
Human lactoferrin inhibits growth of solid tumors and develop- in the Study of Neoplasms. Academic Press, New York and
ment of experimental metastases in mice. Cancer Res. 54, 2310– London.
2312. 33. Legrand, D., Mazurier, J., Metz-Boutigue, M., Jolles, J., Jolles, P.,
14. Sawatzki, G. & Rich, I.N. (1989) Lactoferrin stimulates colony Montreuil, J. & Spik, J. (1984) Characterization and localization
stimulating factor production in vitro and in vivo. Blood Cells 15, of an iron-binding 18-kDa glycopeptide isolated from the
371–385. N-terminal half of human lactotransferrin. Biochim. Biophys. Acta
15. Gahr, M., Speer, C.P., Damerau, B. & Sawatzki, G. (1991) 787, 90–96.
Influence of lactoferrin on the function of human polymorpho- 40. Hakasson, A., Zhivotovsky, B., Orrenius, A., Sabharwal, H. &
nuclear leukocytes and monocytes. J. Leukocyte Biol. 49, 427–433. Svanborg, C. (1995) Apoptosis induced by a human milk protein.
16. De Sousa, M., Breedvelt, F., Dynesius-Trentham, R., Trentham, Proc. Nat. Acad. Sci. USA 92, 8064–8068.
D. & Lum, J. (1988) Iron, iron-binding proteins and immune 34. Zhivotovsky, B., Wade, D., Gahm, A., Orrenius, S. & Nicotera, P.
system cells. Ann. NY Acad. Sci. 526, 310–322. (1994) Formation of 50 kbp chromatin fragments in isolated liver
17. Amouric, M., Marvaldi, J., Pichon, J., Bellot, F. & Figarella, C. nuclei is mediated by protease and endonuclease activation. FEBS
(1984) Effect of lactoferrin on the growth of a human colon Lett. 351, 150–154.
adenocarcinoma cell line-comparison with transferrin. In Vitro 20, 35. Vlassov, A., Florentz, C., Helm, M., Naumov, V., Buneva, V.,
543–548. Nevinsky, G. & Giege, R. (1998) Characterization and selectivity
18. Garre, C., Bianchi-Scarra, G., Sirito, M., Musso, M. & of catalytic antibodies from human serum with RNase activity.
Ravazzolo, R. (1992) Lactoferrin binding sites and nuclear loca- Nucleic Acids Res. 26, 5243–5250.
lization in K562 (S) cells. J. Cell Physiol. 153, 477–482. 36. Nevinsky, G.A., Kit, Yu, Ya., Semenov, D.V. & Buneva, V.N.
19. Arnold, R.R., Cole, M.F. & McGhee, F.M. (1977) A bactericidal (1998) Secretory immunoglobulin A from human milk catalyzes
effect for human lactoferrin. Science 197, 263–265. milk protein phosphorylation. Appl. Biochem. Biotechnol. 75,
20. Yi, M., Kaneko, S., Yu, D.V. & Marakami, S. (1977) Hepatitis C 77–91.
virus envelope proteins bind lactoferrin. J. Virol. 71, 5997–6002. 37. Akagi, K., Murai, K., Hirao, N. & Yamanaka, M. (1976) Puri-
21. Ellison, R.T., Giehl, T.J. & Laforse, F.M. (1988) Damage of the fication and properties of alkaline ribonuclease from human
outer membrane of enteric gram-negative bacteria by lactoferrin serum. Biochim. Biophys. Acta 442, 368–378.
and transferring. Infect. Immun. 56, 2774–2780. 38. De, I., Cardayre, S.B. & Raines, R.T. (1995) A residue to residue
22. Semenov, D.V., Kanyshkova, T.G., Buneva, V.N. & Nevinsky, hydrogen bond mediates the nucleotide specificity of ribo-
G.A. (1998) Interaction of human milk lactoferrin with ATP. nuclease A. J. Mol. Biol. 252, 328–336.
Biochemistry (Mosc) 63, 67–75. 39. Gudding, R. (1979) DNases in milk and blood sera from different
23. Semenov, D.V., Kanyshkova, T.G., Buneva, V.N. & Nevinsky, species. Acta Vet. Scand. 20, 404–416.
G.A. (1999) Human milk lactoferrin binds ATP and dissociates 41. Schwartz, L.M. & Osborne, B.A. (1993) Programmed cell death,
into monomers. Biochem. Mol. Biol. Int. 47, 177–184. apoptosis and killer genes. Immunol. Today 14, 582–590.
24. Kanyshkova, T.G., Semenov, D.V., Buneva, V.N. & Nevinsky, 42. Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis
G.A. (1999) Human milk lactoferrin binds two DNA molecules is associated with endogenous endonuclease activation. Nature
with different affinities. FEBS Lett. 451, 235–237. (London) 284, 555–556.
25. Nevinsky, G.A., Kanyshkova, T.G., Semenov, D.V., Vlassov, 43. Legrand, D., Salmon, V., Coddeville, B., Benaissa, M., Plancke,
A.V., Gal’vita, A.V. & Buneva, V.N. (2000) Secretory immuno- Y. & Spik, G. (1995) Structural determination of two N-linked
globulin A from healthy human mothers’ milk catalyzes nucleic glycans isolated from recombinant human lactoferrin expressed in
acid hydrolysis. Appl. Biochem. Biotechnol. 83, 115–130. BHK cells. FEBS Lett. 365, 57–60.
26. Perret, V., Garcia, A., Puglisi, J.D., Grosjean, H. & Ebel, J.-P. 44. Anderson, B.F., Baker, H.M., Norris, G.E., Rumball, S.V. &
(1990) Conformation in solution of yeast tRNA (Asp) transcripts Baker, E.N. (1990) Apolactoferrin structure demonstrates ligand-
deprived of modified nucleotides. Biochimie 72, 735–743. induced conformational change in transferrins. Nature (London)
27. Vlassov, V.V., Zuber, G., Felden, B., Behr, J.-P. & Giege, R. 344, 784–787.
(1995) Cleavage of tRNA with imidazole and spermine imidazole 45. Kanyshkova, T.G., Buneva, V.N. & Nevinsky, G.A. (2001)
constructs: a new approach for probing RNA structure. Nucleic Lactoferrin and its biological functions. Biochemistry (Mosc) 66,
Acids Res. 23, 3161–3167. 1–7.
28. Savel’ev, E.V., Eneyskaya, K.A., Shabalin, M.V., Filatov, M.V. & 46. Nevinsky, G.A., Favorova, O.O. & Buneva, V.N. (2002) Natural
Neustroev, K.N. (1999) Antibodies with amylolytic activity. catalytic antibodies: new characters in the protein repertoire.
Protein Peptide Lett. 6, 179–184. In Protein–Protein Interactions: A Molecular Cloning Manual
29. Kanyshkova, T.G., Semenov, D.V., Khlimankov, Yu, D., (Golemis, E., ed.), pp. 523–534. Cold Spring Harbor Laboratory
Buneva, V.N. & Nevinsky, G.A. (1997) DNA-hydrolyzing activity Press, New York.