Molecular Characterization of α–Lactalbumin Folding Variants That Induce Apoptosis in Tumor Cells
1999; Elsevier BV; Volume: 274; Issue: 10 Linguagem: Inglês
10.1074/jbc.274.10.6388
ISSN1083-351X
AutoresMalin Svensson, Hemant Sabharwal, Anders Håkansson, Ann‐Kristin Mossberg, Peter Lipniunas, Hakon Leffler, Catharina Svanborg, Sara Linse,
Tópico(s)Proteins in Food Systems
ResumoThis study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk α-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of α-lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule α-lactalbumin showed that the active fraction contains oligomers of α-lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve α-lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of α-lactalbumin. This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk α-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of α-lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule α-lactalbumin showed that the active fraction contains oligomers of α-lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve α-lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of α-lactalbumin. Human milk provides the newborn child with exquisite nutrition, and a mucosal immune system. Breastfeeding protects against respiratory and gastrointestinal infections, due to the presence in milk of molecules with anti-microbial activity: antibodies, potentially bactericidal molecules like lysozyme and lactoferrin (1Lönnerdal B. Forsum E. Am. J. Clin. Nutr. 1985; 41: 113-120Crossref PubMed Scopus (65) Google Scholar), fatty acids that lyse bacteria and viral particles (2Redhead K. Hill T. Mulloy B. FEMS Microbiol. Lett. 1990; 70: 269-274Google Scholar, 3Sarkar N.H. Charney J. Dion A. Moore D. Cancer Res. 1973; 33: 626-629PubMed Google Scholar), and glycoconjugates that inhibit bacterial adherence to epithelial cells (4Svanborg C. Aniansson G. Mestecky J. Sabharwal H. Wold A. Adv. Exp. Med. Biol. 1991; 310: 167-171Crossref PubMed Scopus (7) Google Scholar, 5Andersson B. Dahmén J. Freijd T. Leffler H. Magnusson G. Noori G. Svanborg-Edén C. J. Exp. Med. 1983; 158: 559-570Crossref PubMed Scopus (186) Google Scholar). Epidemiological studies have shown that breastfeeding protects also against cancer (6Davis M.K. Savitz D.A. Graubard B.I. Lancet. 1988; ii: 365-368Abstract Scopus (160) Google Scholar), suggesting that milk contains molecules with anti-tumor activity. We recently observed that a protein fraction of human milk induces apoptosis in tumor cells but not in mature healthy cells (7Håkansson A. Zhivotovsky B. Orrenius S. Sabharwal H. Svanborg C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8064-8068Crossref PubMed Scopus (341) Google Scholar). Surprisingly, the main protein constituent of this fraction was α-lactalbumin. Monomeric α-lactalbumin is secreted by the mammary epithelium and is the major whey protein of human milk (8Heine W.E. Klein P.D. Reeds P.J. J. Nutr. 1991; 121: 277-283Crossref PubMed Scopus (147) Google Scholar). Its main known function is to change the acceptor specificity of β-galactosyltransferase from GlcNAc to Glc, thus enabling the synthesis of lactose in milk (9Brew K. Grobler J. Fox P. Advanced Dairy Chemistry 1. Elsevier, London1992: 191-229Google Scholar, 10Hill R. Brew K. Adv. Enzymol. Relat. Mol. Biol. 1975; 43: 411-490PubMed Google Scholar). The crystal structure of α-lactalbumin has been solved (11Acharya K.R. Ren J.S. Stuart D.I. Phillips D.C. Fenna R.E. J. Mol. Biol. 1991; 221: 571-581Crossref PubMed Scopus (214) Google Scholar) (Fig.1). It is a metalloprotein with high affinity for Ca2+ and other divalent cations (12Aramini J.M. Hiraoki T. Grace M.R. Swaddle T.W. Chiancone E. Vogel H.J. Biochim. Biophys. Acta. 1996; 1293: 72-82Crossref PubMed Scopus (16) Google Scholar, 13Ren J. Stuart D.I. Acharya K.R. J. Biol. Chem. 1993; 268: 19292-19298Abstract Full Text PDF PubMed Google Scholar), and Ca2+ is essential for the folding and structural stability of α-lactalbumin (14Musci G. Berliner L. Biochemistry. 1985; 24: 6945-6948Crossref PubMed Scopus (54) Google Scholar, 15Rao K.R. Brew K. Biochem. Biophys. Res. Commun. 1989; 163: 1390-1396Crossref PubMed Scopus (58) Google Scholar). At low pH, α-lactalbumin forms a relatively stable protein folding variant (16Wu L.C. Schulman B.A. Peng Z.Y. Kim P.S. Biochemistry. 1996; 35: 859-863Crossref PubMed Scopus (61) Google Scholar). This form, the molten globule, has native-like secondary structure but less well defined tertiary structure, and larger stokes radius (17Dolgikh D. Gilmanshin R. Brazhnikov E. Bychkova V. Semisotnov G. Venyaminov S. Ptitsyn O. FEBS Lett. 1981; 136: 311-315Crossref PubMed Scopus (593) Google Scholar). Similar states are formed at elevated temperatures, by reduction of disulfide bonds or by removal of calcium at neutral pH (18Kuwajima K. Proteins Struct. Funct. Genet. 1985; 6: 87-103Crossref Scopus (1483) Google Scholar, 19Kuwajima K. FASEB J. 1996; 10: 102-109Crossref PubMed Scopus (434) Google Scholar, 20Pfeil W. Biochim. Biophys. Acta. 1987; 911: 114-116Crossref PubMed Scopus (27) Google Scholar). Unlike monomeric α-lactalbumin from whey, the apoptosis-inducing component was purified from the casein fraction (precipitated at pH 4.3), and behaved as a multimeric protein rather than a monomer. Furthermore, native monomeric α-lactalbumin from human milk whey was inactive in the apoptosis assay. These observations suggested that the active fraction contains an alternative molecular form of α-lactalbumin. Here we show, using mass spectrometry, gel filtration, UV absorption, fluorescence, and CD spectroscopy, that the apoptosis-inducing form of α-lactalbumin is a mixture of monomeric and multimeric forms with molten globule-like properties and suggest that folding variants of α-lactalbumin differ in biologic activity. ANS 1The abbreviations used are: ANS, 8-anilinonaphtalene-1-sulfonic acid; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; MALDI-TOF, matrix assisted laser desorbtion ionization-time of flight mass spectrometry; PBS, phosphate-buffered saline.ammonium salt was from Fluka, Buchs, Switzerland. Ammonium sulfate, Tris, calcium chloride, HCl, sodium chloride, methanol, acetic acid, glycine, sodium barbitone, acetonitrile, trifluoroacetic acid, sinapinic acid, and potassium phosphate were from Merck, Darmstadt, Germany. EDTA, SDS, bromphenol blue, and glycerol were from Sigma. Potassium oxalate was from Riedel-de Haen, Seelze, Germany. Agarose (Sea Kem GTG) was from Bioproducts, Rockland, MI. PAGE ready gels were from Bio-Rad. RPMI 1640 cell culture media, fetal calf serum, nonessential amino acids, sodium pyruvate. and gentamicin were from Life Technologies, Paisley, United Kingdom. The Biotin Labeling kit was from Boehringer Mannheim, GmbH, Germany. All chemicals were of the highest grade commercially available. Native, monomeric α-lactalbumin was purified from human milk by ammonium sulfate precipitation. The ammonium sulfate was added as a salt, 264 g/liter milk, and the mixture was incubated overnight at 4 °C. The mixture was centrifuged (Sorvall RC-5B refrigerated superspeed centrifuge, Du Pont Instruments, Wilmington, DE) at 5000 × g for 15 min. The whey fraction was collected, lyophilized, and dissolved in 50 mmTris/HCl with 35 mm EDTA, pH 7.5. A 400-ml phenyl-Sepharose column (Pharmacia Biotech, Uppsala, Sweden) was packed in 50 mm Tris/HCl with 1 mm EDTA, pH 7.5, 25 °C and a 500-ml sample was loaded onto the column. The column was first washed with 50 mm Tris/HCl with 1 mm EDTA, pH 7.5, and α-lactalbumin was then eluted from the column with 50 mm Tris/HCl with 1 mm CaCl2, pH 7.5, thus yielding the native, Ca2+ bound form of α-lactalbumin. The classical molten globule state was obtained by lowering the pH of a solution of native monomeric α-lactalbumin to 2.0 by adding 0.1m HCl. This material was used as a control sample in subsequent spectroscopic studies. In the cellular experiments, EDTA (0.14 mm/mg) was added to α-lactalbumin to remove Ca2+ and thus form the apo state, which is molten globule-like. Frozen human milk was thawed and centrifuged (Sorvall RC-5B refrigerated superspeed centrifuge, Du Pont Instruments) at 2500 × g for 15 min; the upper fat layer was removed. Casein was isolated by an overnight incubation at 4 °C with 10% potassium oxalate followed by a second overnight incubation at 4 °C after lowering the pH to 4.3 using 1m hydrochloric acid and heating the solution to 32 °C for 2 h. The casein precipitate was harvested by centrifugation at 5000 × g for 15 min, washed by 3–5 cycles of centrifugation, and resuspension in distilled water and lyophilized. Casein was fractionated on DEAE-Trisacryl M (Biosepra, Villeneuve la Garenne, France) using an FPLC instrument (Pharmacia Biotech Inc.) with increasing NaCl gradient. The sample was loaded in buffer A (0.01 m Tris-HCl, pH 8.5) at 25 °C and eluted by increasing proportions of buffer B (buffer A containing 1 m NaCl). Gradient program: start 15% B; from 0 to 60 ml: linear gradient from 60 to 90 ml: 30% B at 90 ml: 100% B; for 10 min; thereafter 100% A. Flow rate: 1 ml/min, recorder: 0.1 cm/min. The buffers were degassed and filtered through 0.22-μm filters before use. The peaks were monitored at 280 nm, and the fraction size was 3 ml. The eluate was desalted by dialysis (Spectra/Por, Spectrum Medical Industries, Laguna Hills, CA, membrane cut off 3.5 kDa) against distilled water for at least 48 h and lyophilized. The active fraction from anion exchange chromatography was subjected to size exclusion separation on Sephadex G-50 column (Pharmacia Biotech, Uppsala, Sweden, 93 × 2.5 cm) equilibrated with 0.06 m sodium phosphate buffer, pH 7.0, 25 °C. The flow rate was 0.5 ml/min, peaks were monitored at 280 nm and 3-ml fractions were collected and pooled. The pools were desalted by dialysis (membrane cut off 3.5 kDa) against distilled water for at least 48 h and lyophilized before further analysis in subsequent experiments. Further gel filtration of the active fraction from the anion exchange chromatography was performed on a Superose 12 column (Pharmacia Biotech, 30 × 1.0 cm) in 10 mm Tris/HCl, pH 7.5, 25 °C with 0.15 m NaCl. The flow rate was 0.3 ml/min, the fraction size was 0.5 ml and peaks were monitored at 280 nm. Observed peaks were collected, desalted by dialysis against distilled water, and lyophilized. Analytical PAGE was performed using 4–20% polyacrylamide precast gels on a Bio-Rad Mini Protean II cell. To 10 μl of the lyophilized fractions from anion exchange chromatography or gel filtrations dissolved in distilled water (5–10 mg/ml), an equal volume of sample buffer (13.1% 0.5 m Tris-HCl, pH 6.8, 10.5% glycerol, and 0.05% bromphenol blue) was added. Samples (20 μl) were then loaded onto the gel, which was run in Tris glycine buffer, pH 8.3, with 0.1% SDS at 200 V constant voltage for 40 min. Proteins were stained by immersing the gel in 0.1% Coomassie Blue solution in water/methanol/acetic acid (5:4:1) for 0.5 h. Destaining was by several changes in 40% methanol, 10% acetic acid until a clear background was obtained. After PAGE, the protein bands of peak K from the G-50 column were transferred by Western blotting onto polyvinylidene difluoride membranes. The protein bands were visualized by Coomassie Blue staining and the stained bands were cut out for protein sequencing. Protein sequencing was also performed on an aliquot of each of the peaks 1–4 from the Superose-12 column directly. All samples were subjected to Edman degradation performed in an automated pulse-liquid sequencer (Applied Biosystems model 477A). Peak K was analyzed on a VG Bio-Q ESI-MS (Fisons/VG, Manchester, UK) equipped with an atmospheric pressure electrospray ion source and a quadruple mass analyzer with a maximum mass range of 4000. The mass spectrometer was scanned from m/z 600 to 2000 in 10 s. The mass resolution was set to 500. The data system was operated as a multichannel analyzer and 5 scans were averaged to obtain the final spectrum. The electrospray carrier solvent was 1% acetic acid in acetonitrile/water, 1:1, and the flow rate was 2–4 μl/min. The sample was dissolved at a concentration of 10–20 pmol/μl in the carrier solvent and 5 μl was injected. The molecular weight of sample components was estimated from the m/z values of series of ions as described earlier. Peak K was analyzed by MALDI mass spectrometry on an LDI 1700 time of flight mass spectrometer equipped with a pulsed nitrogen laser (337 nm) (Biomolecular Separations Inc., Reno, NE). The laser power was set to 8.6 microjoule and the spectrum was the sum of 140 laser shots. Sinapinic acid was used as a matrix and bovine serum albumin was used as the external standard. About 100 μg of the protein was dissolved in 50 μl of water and 0.1% trifluoroacetic acid. 10 μl of this solution was mixed with 10 μl of 50 mm sinapinic acid. The probe was loaded with 0.8 μl of the sample mixture, vacuum dried, loaded with another 0.8 μl of sample, and vacuum dried again before being inserted into the mass spectrometer. Prior to spectroscopic analysis, the proteins or protein fractions were dialyzed against doubly distilled water and lyophilized. Stock solutions of each sample were prepared by dissolving the lyophilized material in 10 mm potassium phosphate buffer at pH 7.5. The concentrations of the stock solutions were determined using amino acid analysis after acid hydrolysis. The spectra were recorded on solutions prepared by diluting aliquots of stock solution into 10 mm potassium phosphate buffer at pH 7.5. All spectra were recorded at 25 °C. UV absorbance spectra were recorded at room temperature on a GBC UV/VIS 920 spectrophotometer, in a quartz cuvette with 1-cm path length. Fluorescence spectra were recorded at 25 °C on a Perkin-Elmer LS-50B spectrometer using a quartz cuvette with 1-cm excitation path length. Intrinsic (tryptophan) fluorescence emission spectra were recorded between 305 and 530 nm (step 1 nm) with excitation at 295 nm. The excitation band width was 3 nm and the emission band width was 5 nm. ANS fluorescence emission spectra were recorded between 400 and 600 nm (step 1 nm) with excitation at 385 nm. Both the excitation and emission bandpass were set to 5 nm. Circular dichroism (CD) spectra were obtained using a JASCO J-720 spectropolarimeter with a JASCO PTC-343 Peltier-type thermostated cell holder. Quartz cuvettes were used with 1-cm path length in the near UV range and 1 and 0.1-mm path length in the far UV range. Near UV spectra were recorded between 320 and 240 nm, and far UV spectra between 250 and 182 nm. The wavelength step was 1 nm, the response time was 4 s, and the scan rate was 10 nm/min. Six scans were recorded and averaged for each spectrum. Baseline spectra were recorded with pure buffer in each cuvette and subtracted from the protein spectra. The mean residue ellipticity θm (mdeg × cm2 × dmol−1) was calculated from the recorded ellipticity, θ, as θm = θ/(c·n·l), where c is the protein concentration in M, n the number of residues in the protein (123 in this case), l the path length in m, and θ the ellipticy in degrees. The L1210 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA), cultured in 25-cm2 flasks (Falcon, Becton Dickinson, NJ) in RPMI 1640 supplemented with 10% fetal calf serum, nonessential amino acids, sodium pyruvate, 50 μg of gentamicin/ml, kept at 37 °C in a humidified atmosphere containing 5% CO2, with change of medium every 3 days. The cells were harvested from the culture flasks by centrifugation (200 × g for 10 min). The cell pellet was resuspended in medium and seeded into 24-well plates, 2 × 106/well (Falcon, Becton Dickinson, NJ). Cells were exposed to the different forms of α-lactalbumin, with medium as a control. At time 0, 100 μl of medium was aspirated from each well, replaced by 100 μl of the different experimental solutions and incubated at 37 °C in an atmosphere of 5% CO2 for 6 h. Cells were harvested from the 24-well plates by aspiration, resuspended in PBS (5 ml), washed, and resuspended in 1 ml of PBS. For analysis, 30 μl of the washed cell suspension was mixed with 30 μl of a 0.2% trypan blue solution and the number of stained cells (dead cells) per 100 cells was determined by interference contrast microscopy (Ortolux II, Leitz Wetzlar, Germany). Oligonucleosome length DNA fragments were detected by agarose gel electrophoresis. The remainder of the washed cell suspension (970 μl, 2 × 106/ml) was lysed in 5 mm Tris, 20 mm EDTA, 0.5% Triton X-100, pH 8.0, at 4 °C for 1 h and centrifuged at 13,000 ×g for 15 min. DNA was ethanol precipitated overnight in −20 °C, treated with RNase proteinase K, and loaded on 1.8% agarose gels, and electrophoresed with constant voltage set at 50 V overnight. DNA fragments were visualized with ethidium bromide using a 305-nm UV-light source and photographed using Polaroid type 55 positive-negative film. L1210 cell suspension (4 × 106 cells/ml, 95 μl) were incubated at room temperature with 5 μl of biotinylated fraction VI or native monomeric α-lactalbumin (5 mg/ml, biotinylated according to the manufacturer's instructions), and then washed in PBS with centrifugation at 320 × g for 10 min to remove unbound protein. To detect intracellular protein, cells exposed to biotinylated protein were permeabilized with saponin to allow entry of fluorescein isothiocyanate-conjugated streptavidin. Cells harvested by centrifugation at 320 × g were fixed by suspension in phosphate-buffered paraformaldehyde (4%) (21Sander B. Andersson J. Andersson U. Immunol. Rev. 1991; 119: 65-92Crossref PubMed Scopus (415) Google Scholar) for 5 min at room temperature, washed in PBS, and permeabilized with 0.1% saponin in PBS. After washing in 0.1% saponin, fluorescein isothiocyanate-conjugated streptavidin (1:100 in 0.1% saponin, 100 μl) was added and the cells were incubated for 30 min at room temperature. The cells were washed twice in PBS/saponin and once in PBS, mounted on a glass slide and analyzed in a Bio-Rad 1024 laser scanning confocal equipment (Bio-Rad, Hemel-Hempstead, UK) attached to a Nikon Diaphot inverted microscope (Nikon, Japan). Human milk samples from four donors were separated into casein and whey and the fractions were tested for apoptosis induction in L1210 leukemia cells. The activity precipitated with casein and further purification was from the casein fraction. Following anion-exchange chromatography, six fractions were collected and tested for apoptosis induction. Fractions I-V completely lacked activity. Fraction VI that eluted with 1 m NaCl contained all of the apoptosis inducing activity (Fig.3 A). PAGE of fraction VI revealed one major band in the 14-kDa region, and additional bands in the molecular mass range of 30, 60, and 100 kDa (Fig. 2 A, inset). After heat treatment (100 °C, 5 min), fraction VI lost its activity (data not shown).Figure 2Anion exchange chromatography and gel filtration. Panel A, ion-exchange chromatogram of human casein. 30 mg of casein was fractionated using a DEAE-Trisacryl column attached to a FPLC system using a stepwise NaCl gradient, at 25 °C. The apoptosis-inducing fraction eluted after 1 m NaCl indicated by the arrow. Inset, PAGE of the apoptosis inducing fraction VI and monomeric α-lactalbumin (ALA). The molecular weight standards (lane S) are: myosin (M r = 200,000), β-galactosidase (M r = 116, 250), phosphorylase b(M r = 97, 400), bovine serum albumin (M r = 66, 200), ovalbumin (M r = 45,000), carbonic anhydrase (M r = 31,000), soybean trypsin inhibitor (M r = 21,500), and lysozyme (M r = 14,400), respectively. Panel B,size exclusion chromatogram of fraction VI from the ion-exchange column. 100 mg was applied to a Sephadex G-50 column (93 × 2.5 cm) equilibrated with 0.06 m sodium phosphate buffer, pH 7.0, 25 °C. The flow rate was 30 ml/h and the fraction size was 3 ml. Two well separated peaks were obtained. Inset, PAGE of peaks K and L. The molecular weight standards are as in panel A. Panel C, size exclusion chromatogram of fraction VI on a Superose 12 column. Fraction VI (10 mg) was suspended in 500 μl of 10 mm potassium phosphate buffer, pH 7.5, 25 °C (flow rate 0.3 ml/min, fraction size 0.5 ml). Peak 1 eluted with the void volume (8 ml), peak 2 eluted at 9.4–11.6 ml corresponding to a molecular mass of 150–400 kDa, peak 3 eluted at 13.8–14.2 ml corresponding to a mass of 30–42 kDa, and peak 4 eluted at 15.0–15.2 ml, corresponding to monomeric α-lactalbumin, 14 kDa.Inset, PAGE of peaks 1–4, monomeric α-lactalbumin (ALA) and fraction VI. The molecular weight standards (S) are as in panel A.View Large Image Figure ViewerDownload (PPT)Figure 2Anion exchange chromatography and gel filtration. Panel A, ion-exchange chromatogram of human casein. 30 mg of casein was fractionated using a DEAE-Trisacryl column attached to a FPLC system using a stepwise NaCl gradient, at 25 °C. The apoptosis-inducing fraction eluted after 1 m NaCl indicated by the arrow. Inset, PAGE of the apoptosis inducing fraction VI and monomeric α-lactalbumin (ALA). The molecular weight standards (lane S) are: myosin (M r = 200,000), β-galactosidase (M r = 116, 250), phosphorylase b(M r = 97, 400), bovine serum albumin (M r = 66, 200), ovalbumin (M r = 45,000), carbonic anhydrase (M r = 31,000), soybean trypsin inhibitor (M r = 21,500), and lysozyme (M r = 14,400), respectively. Panel B,size exclusion chromatogram of fraction VI from the ion-exchange column. 100 mg was applied to a Sephadex G-50 column (93 × 2.5 cm) equilibrated with 0.06 m sodium phosphate buffer, pH 7.0, 25 °C. The flow rate was 30 ml/h and the fraction size was 3 ml. Two well separated peaks were obtained. Inset, PAGE of peaks K and L. The molecular weight standards are as in panel A. Panel C, size exclusion chromatogram of fraction VI on a Superose 12 column. Fraction VI (10 mg) was suspended in 500 μl of 10 mm potassium phosphate buffer, pH 7.5, 25 °C (flow rate 0.3 ml/min, fraction size 0.5 ml). Peak 1 eluted with the void volume (8 ml), peak 2 eluted at 9.4–11.6 ml corresponding to a molecular mass of 150–400 kDa, peak 3 eluted at 13.8–14.2 ml corresponding to a mass of 30–42 kDa, and peak 4 eluted at 15.0–15.2 ml, corresponding to monomeric α-lactalbumin, 14 kDa.Inset, PAGE of peaks 1–4, monomeric α-lactalbumin (ALA) and fraction VI. The molecular weight standards (S) are as in panel A.View Large Image Figure ViewerDownload (PPT) Fraction VI was applied on a Sephadex G-50 column. Two well separated peaks (K andL, Fig. 2 B) were obtained. Peak K eluted near the void volume and contained all of the apoptotic activity, peak L eluted at the position of a 14-kDa protein and had no effect on cell viability. PAGE of peak K showed one major band at about 14 kDa, and additional bands of about 30, 60, and 100 kDa. Peak L gave only one band at 14 kDa (Fig. 2 B, inset). The different bands of peak K were subjected to N-terminal amino acid sequence analysis (Table I). The first 30 residues of the band at 14 kDa and the first 9 residues of the 30-kDa band were identical to the N-terminal sequence of human α-lactalbumin, except for residue 6, which was not detected. The main N-terminal sequence of the 60-kDa band and the two 100-kDa bands was also identical to human α-lactalbumin, but some sequencing cycles showed heterogeneity. These results suggested that peak K contained α-lactalbumin complexes of increasing molecular sizeTable IN-terminal amino acid sequence of the protein bands of peaks K and 1–4 and human α-lactalbumin (HLA)SequencesHLA14 kDaLys-Gln-Phe-Thr-Lys-Cys-Glu-Leu-Ser-Gln--Leu-Leu-Lys-Asp-Ile-Asp-Gly-Tyr-Gly-Gly--Ile-Alα-Leu-Pro-Pro-Leu-Ile-Asp-Thr-Met-Peak K14 kDaLys-Gln-Phe-Thr-Lys-Unk-Glu-Leu-Ser-Gln--Leu-Leu-Lys-Asp-Ile-Asp-Gly-Tyr-Gly-Gly--Ile-Alα-Leu-Pro-Pro-Leu-Ile-Asp-Thr-Met-30 kDaLys-Gln-Phe-Thr-Lys-Unk-Glu-Leu-Ser-Gln-60 kDaLys-Gln-Phe-Leu-Lys-Arg Pro Lys Thr Pro100 kDaLys-Gln-Phe-Thr-Unk-Unk-Glu-Leu-Unk-Gln-Asn Ile Ser ValTyr AsnPeak 1Lys-Gln-Phe-Thr-Lys-UnkPeak 2Lys-Gln-Phe-Thr-Lys-UnkPeak 3Lys-Gln-Phe-Thr-Lys-UnkPeak 4Lys-Gln-Phe-Thr-Lys-UnkUnk, indicates unknown; according to published results, residue 6 in α-lactalbumin is cysteine. Residues shown below the 60- and 100-kDa sequence of peak K are other possible candidates. Open table in a new tab Unk, indicates unknown; according to published results, residue 6 in α-lactalbumin is cysteine. Residues shown below the 60- and 100-kDa sequence of peak K are other possible candidates. The effect on cell viability and induction of DNA fragmentation was compared between the different milk fractions (Fig. 3 A). A rapid reduction of cell viability from >95% to 95% pure. Peaks 1, 2, and 3 showed a higher background (steps 1–10), but no dominating second sequence suggestive of another protein (Table I). Fraction VI and peaks 1–4 from the Superose-12 column were analyzed by spectroscopic techniques. Monomeric α-lactalbumin in the native or the acid-induced molten globule state were included as controls. By UV absorption spectroscopy α-lactalbumin and fraction VI showed virtually identical spect
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