Towards large-scale purification of natural CSF-1 from human placenta tissue extracts

The monocyte-macrophage colony-stimulating factor (colony-stimulating factor 1) is characterized and partially purified from industrially processed human tissues for the first time. A five-step purification procedure using placenta tissue extracts furnished a 13,620-fold enrichment of biological activity. This procedure includes a "pilot" scale anion-exchange chromatography at pH 4.5, gel permeation, and lectin affinity separation followed by HPLC steps (hydrophobic interaction and C18 reverse-phase chromatographies). The purified bioactive material, which stimulates only monocyte-macrophage progenitors and mature cells, showed an Mr of 58,000-62,000 (gel filtration) and an isoelectric point of 3.8-4.0. The hydrophobicity of the molecule was low, and the biological activity was eluted at 50% acetonitrile on a C18 reverse-phase HPLC column. It was totally inactivated by 2-beta-mercaptoethanol reduction and heat treatment. Immunoprecipitation and neutralization of biological activity with specific anti-CSF-1 antibodies (not shown) demonstrated that this material was CSF-1. Step 5 of this protocol yielded two silver-stained bands on 12.5% SDS-PAGE: a major 55-kDa band (96%) and a minor 33-kDa band (4%). CSF-1 was detected exclusively in a band of 52-62 kDa by both Western immunoblotting and bioassays. Immunoaffinity techniques using antibodies directed against selective epitopes on the placental CSF-1 are now considered to purify this material to homogeneity. This approach to the mass production of natural CSF-1 from human tissue has advantages with respect to both the difficulty of post-translational processing of bioactive material in procaryotes and the cost of eucaryotic cell cultures.     

Characterization of deglycosylated human urinary colony-stimulating factor (CSF-1)

1. Isolation of CSF-1 from human urine was performed through five purification steps. These include concentration by dialysis, silica gel absorption, hydrophobic chromatography and phenyl-Sepharose CL-6B, Fast Protein Liquid Chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. These methods have been reported in a previous paper (Tao et al., 1987). 2. The isolated CSF-1 which exhibits a one band pattern on SDS-PAGE under non-reducing conditions after Coomassie Blue and silver stainings. CSF-1 was purified 100,000-fold and has a specific activity of 2.16 x 10(7) units/mg protein. Its apparent Mr is 57,000 with an isoelectric point pI = 5.8-6.0 CSF-1 is a glycoprotein with 40% of carbohydrate (w/w). 3. An almost complete removal of the carbohydrate moiety from CSF-1 was obtained after treatment with trifluoromethanesulfonic (TFMS) acid followed by gel filtration on Sephadex G-25 (Fine). The deglycosylated (DG) CSF-1 possesses an apparent Mr of 38,000 and an isoelectric point, pI: 6.2 as compared to native-CSF-1 (N-CSF-1), Mr = 57,000 and pI = 5.8 respectively. 4. The TFMS treatment did not alter the activities of CSF-1 as shown by biological assay and receptor binding assay. The thermostability experiment revealed that DG-CSF-1 was less stable than N-CSF-1. The circular dichroism spectra (CD) of N-CSF-1 and DG-CSF-1 were different. 5. The features of interaction of iodinated-N-CSF-1 and iodinated-DG-CSF-1 with single cell suspensions from human peritoneal macrophage were studied. The binding activity of peritoneal macrophage was the highest among all cells examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of human urinary colony-stimulating factor

CSF-1 was isolated from a large volume of human normal urine (10,000 l), using the following 5 stages of purification: concentration by dialysis, silica gel adsorption, hydrophobic chromatography on phenyl-Sepharose CL-6B, fast protein liquid chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. We have isolated 8 mg of purified CSF-1 which migrated as a single band under non-reducing conditions in SDS-PAGE (staining with Coomassie Blue and the sensitive silver techniques). But in the presence of dithiothreitol, the SDS-PAGE pattern revealed a minor second band with a molecular mass of 50,000 Da. CSF-1 was purified 100,000-fold and has a specific activity of 2.16 X 10(7) units/mg protein. Its apparent molecular mass is 57,000 Da with an isoelectric point, pI = 5.8-6.0. The amino-acid composition is reported and compared with that of murine CSF-1. The carbohydrate content (sialic acid, sulphate groups, N-acetylglucosamine, N-acetylgalactosamine) was also determined, and it shows that CSF is a glycoprotein.     

Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells

Sex-specific agglutinins from the cell surface of haploid cells of Saccharomyces cerevisiae (X2180, mt^a and mt) were purified and analysed. The constitutive agglutinin from mt^a cells was extractable with 3 mM dithiothreitol. It was shown to be a glycoprotein (3% mannose) with an apparent Mr of 43,000 based on gel filtration, but in SDS-PAGE it behaved as a much smaller molecule (Mr between 18,000 and 26,000). About one in three amino acids was a hydroxyamino acid. Its biological activity was resistant to boiling for 1 h, but sensitive to pronase. Intact mt cells retained their agglutinability in the presence of dithiothreitol but limited trypsinizing released a biologically active agglutinin fragment. It had an apparent Mr of 320,000 (gel filtration). When analysed by SDS-PAGE, a single diffuse band with an apparent Mr of 225,000 was observed. The protein was 94% (w/w) mannose with a trace of N-acetyl glucosamine. Its biological activity was almost completely lost after boiling for 1 h. Both agglutinins behaved as monovalent molecules and specifically inhibited the biological activity of both noninduced and pheromone-induced cells. Pheromone treatment of mt^a cells resulted in an apparent 32-fold increase in agglutinin activity at the cell surface, whereas pheromone treatment of mt cells only doubled the apparent agglutinin activity.Abbreviations mt mating type - DTT dithiothreitol - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - YPG yeast-peptone-glucose - PAS periodic-acid-Schiff reagent     

Purification and characterization of activated human erythrocyte prolidase

Prolidase (E.C. 3.4.13.9) has been purified 7500-fold to homogeneity from human erythrocytes in a Mn2+-activated form using conventional and fast protein liquid chromatography columns. The procedure includes a 1-h incubation of the crude hemolysate at 50 degrees C with 1 mM MnCl2. Following this novel step, prolidase retains full activity, obviating the requirement for preincubation of each enzyme fraction with Mn2+ prior to assay. Preincubation with MnCl2 does not change the isoelectric point of the enzyme. The molecular weight of the purified enzyme was 58,000 when measured by SDS-PAGE. Western blotting, using rabbit antibody raised to human kidney prolidase, with partially purified erythrocyte enzyme revealed a cross-reacting band at Mr 58,000.     

Characterization of pure human renal renin. Evidence for a subunit structure

Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.     

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.     

Purification and characterization of Leydig cell luteinizing hormone receptor

We have purified the testicular luteinizing hormone (LH/human choriogonadotropin (hCG)) receptor by sequential affinity chromatography on hCG-Sepharose. The purified LH/hCG receptor was identified as a single protein of Mr = 90,000 +/- 2,000 on sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE), showed high affinity binding for hCG, and a binding capacity of 3.8 nmol/mg of protein. Electrophoretically blotted receptor retained the ability to bind 125I-hCG on nitrocellulose membrane, and the Mr of radioactive band was consistent with that revealed by silver staining. Autoradiography after SDS-PAGE analysis of cross-linked purified receptor-hCG complex showed Mr = 145,000 and Mr = 105,000 bands. These results are consistent with a Mr value for the receptor of 90,000 after accounting for contribution by the intact hormone or its alpha-subunit. Analysis of the free receptor by fast protein liquid chromatography on Superose 12 revealed a single peak of binding activity for 125I-hCG which eluted in the position of Mr = 200,000-240,000 in the presence of Triton X-100. Since a single protein species is observed under reducing or nonreducing conditions in SDS-PAGE, the receptor could exist in the membrane as a dimeric form composed of subunits Mr = 90,000 associated through noncovalent interactions. The pure receptor can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (approximately 0.3 mol of phosphate/mol of receptor). This phosphorylation does not affect the binding characteristics of the receptor. The method described is simple and allows rapid purification of microgram amounts of biological active Leydig cell LH/hCG receptor for structural, functional, and immunological studies.     

The human interferon-gamma receptor. Purification, characterization, and preparation of antibodies

The receptor for human interferon-gamma (IFN-gamma) was purified from foreskin fibroblasts. Triton X-100 extracts obtained from either intact cells or membrane preparations were passed through an immobilized interferon-gamma column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of eluted fractions revealed a major band of Mr = 95,000 and minor bands of Mr = 80,000 and 60,000. Further purification was obtained by steric exclusion and by lectin chromatography. The purified receptor retained the ability to bind 125I-IFN-gamma with a Kd of 2.2 X 10(-10) M, a value close to that obtained with intact fibroblasts (5 X 10(-10) M). A complex of Mr = 105,000-125,000 was visualized by immunoprecipitation of 125I-IFN-gamma cross-linked to the purified receptor followed by SDS-PAGE and autoradiography. A similar complex was obtained when 125I-IFN-gamma was cross-linked to intact cells. Immunization of mice with the excised SDS-PAGE band of Mr = 95,000 elicited antibodies that blocked the antiviral activity of IFN-gamma and immunoprecipitated the cross-linked complex of 125I-IFN-gamma and its receptor.     

The specific binding activities of human urinary radioiodinated colony-stimulating factor-1 to various human tissue cells

1. Colony-stimulating factor (CSF-1) was isolated from a large volume of fresh normal human urine by 5 steps of purification and enrichment. 2. The purification factor is 100,000 fold and the purified compound exhibits a 2.16 x 10(7) U/mg of protein sp. act. 3. The isolated CSF-1 is a sialoglycoprotein with 41.5% of carbohydrate. The almost complete removal of this carbohydrate moiety (up to 91%) was achieved by incubation with trifluoromethane sulfonic acid. 4. The deglycosylated CSF-1 (DG-CSF-1) possesses an apparent Mr 38,000 compared to native CSF-1 with an initial Mr 57,000 (Goa et al., 1988). 5. The features of the interaction of radio-iodinated [125I]CSF-1 with single cell suspensions from various human tissues (bone marrow, spleen, blood, peritoneal cavity, alveolar lavage, lymph node and thymus), were studied. 6. The binding activity of peritoneal macrophages was the highest among the cells examined and erythrocytes, thymus and blood granulocytes showed no CSF-1 binding. 7. On incubation with [125I]CSF-1 at 0 degrees C, cellular binding of [125I]CSF-1 reached a stable maximum within 16 hr. This is in contrast to the association behaviour at higher temperature. 8. At 37 degrees C, cellular associated [125I]CSF-1 levels reached, within 90 min, an unstable maximum which was up to 10 times less than that occurring under the same conditions at 0 degree C. From the Scatchard plot analysis, we obtained the affinity constant and the number of receptor(s). 9. The binding site is sensitive to trypsin. 10. The receptor alone, (labelled by cross-linking to [125I]CSF-1 with di-succinylimidyl-suberate), is a polypeptide with an approx. Mr 110,000. 11. Our results showed that the receptor of CSF-1 is a tyrosin-kinase.     

Penicillium chrysogenum extracellular acid phosphatase: purification and biochemical characterization

An extracellular acid phosphatase (EC 3.1.3.2) from crude culture filtrate of Penicillium chrysogenum was purified to homogeneity using high-performance ion-exchange chromatography and size-exclusion chromatography. SDS-PAGE of the purified enzyme exhibited a single stained band at an Mr of approx. 57,000. The mobility of the native enzyme indicated the Mr to be 50,000, implying that the active form is a monomer. The isoelectric point of the enzyme was estimated to be 6.2 by isoelectric focusing. Like acid phosphatases from several yeasts and fungi the Penicillium enzyme was a glycoprotein. Removal of carbohydrate resulted in a protein band with an Mr of 50,000 as estimated by SDS-PAGE, suggesting that 12% of the mass of the enzyme was carbohydrate. The enzyme was catalytically active at temperatures ranging from 20 degrees C to 65 degrees C with a maximum activity at 60 degrees C and the pH optimum was at 5.5. The Michaelis constant of the enzyme for p-nitrophenyl phosphate was 0.11 mM and it was inhibited competitively by inorganic phosphate (ki = 0.42 mM).     

Identification of a low molecular weight form of epithelial transforming growth factor

We have purified a novel form of epithelial transforming growth factor (TGFe) from bovine kidney. Acid ethanol extracts of kidney were fractionated by size exclusion, reverse phase and cation exchange chromatography and activity was monitored by measuring growth of SW13 adrenocortical carcinoma cells in soft agar. The purified material was highly cationic, bound weakly to heparin and gave a band at 13-15000 Mr by SDS-PAGE following Bolton-Hunter iodination. This band correspond to the migration of biological activity extractable from gel slices. The results suggest that we have isolated a truncated form of TGFe which nonetheless retains biological activity.     

Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native alpha 2 beta 2 insulin receptor subunit complex

The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing (0.1% SDS) or nondenaturing (0.1% Triton X-100) conditions. Pretreatment of 32P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the alpha 2 beta 2 insulin receptor complex (Mr 400,000) into the monomeric 95,000 beta subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the alpha 2 beta 2 heterotetrameric complex with essentially no alpha beta heterodimeric or free monomeric beta subunit species present. This suggests that the insulin receptor can reoxidize into the Mr 400,000 complex after the removal of DTT by gel filtration chromatography. Surprisingly, these apparently reoxidized insulin receptors were also observed to be functional with respect to insulin binding, albeit with a 50% decrease in affinity for insulin and insulin stimulation of the beta subunit autophosphorylation. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT followed by incubation with excess N-ethylmaleimide prior to gel filtration chromatography in 0.1% Triton X-100. Under these conditions the insulin receptors migrated as the Mr 400,000 alpha 2 beta 2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

重组人纤溶酶原Kringle1-3的生物学活性鉴定

目的：检测重组人纤溶酶原Kringle1-3（K1-3）的生物学活性。方法：用含重组人纤溶酶原K1-3基因的表达载体pET21a-Angio（K1-3）转化表达宿主菌大肠杆菌BL21（DE3）后诱导表达,表达产物经溶解、复性和纯化后,进行SDS-PAGE,计算其相对分子质量;用BCA法测蛋白浓度,用细胞抑制实验（MTT法）和鸡胚绒毛膜尿囊膜（CAM）实验鉴定纯化产物对血管内皮细胞增殖和血管生成的抑制效果。结果：表达产物的相对分子质量为38000,与预期值一致;细胞抑制实验和CAM实验结果表明表达产物具有特异抑制血管内皮细胞增殖、血管生成的功能。结论：所纯化的重组人纤溶酶原K1-3具有抑制血管内皮细胞的生物学活性,为该蛋白在病理性血管疾病等方面的应用研究提供了材料。     

Isolation, characterization and immunocytochemical localization of bovine trophoblast protein-1

Bovine trophoblast protein-1 (bTP-1) was isolated to 90% purity from culture medium of Day 18-20 conceptuses incubated in vitro (in the presence of L-[3H]leucine) by a combination of Sephacryl S-200 gel filtration chromatography and O-(diethylaminoethyl) (DEAE) anion-exchange high-performance liquid chromatography (DEAE-HPLC). The radiolabeled protein had an Mr of 21,200 +/- 800 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had three isoelectric variants (pI 5.7-6.5) by two-dimensional SDS-PAGE. DEAE-HPLC-enriched bTP-1 cross-reacted with anti-o TP-1 serum on Western blots. A monospecific antiserum against bTP-1 was produced by excising the bTP-1 polypeptide band from preparative SDS-PAGE gels. Antiserum reacted with a single polypeptide with an Mr of 21,200 as determined by Western blotting of SDS-PAGE-separated conceptus medium proteins and by immunoprecipitation from L-[35S]methionine-labeled medium proteins followed by SDS-PAGE and autoradiography. Bovine trophoblast protein-1 was localized by immunocytochemistry in the cytoplasm of both mono- and binuclear trophectoderm cells of Day 20 bovine conceptuses, indicating that it is a product of the trophoblast.     

Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase

Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.     

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

Cysteine proteinase content of rat muscle lysosomes. Evidence for an unusual proteinase activity

Lysosomal cysteine proteinases were fractionated from partially purified rat muscle lysosomes. By gel filtration on Sephadex G75, cathepsin D was separated from two thiol-requiring proteolytic fractions of Mr 25 000 and 55 000, respectively. By chromatofocusing, the first fraction (Mr = 25 000) was resolved into three isoenzymic forms of cathepsin H, eluted at pH 5.8, 6.0 and 7.2, respectively, and two isoenzymic forms of cathepsin B, eluted at pH 5.5 and 5.25. Cathepsin H isoenzymes hydrolyzed Arg-NNap and BANA, were totally inhibited by 1 mM p-CMB and only to 60% by 5.10(-5) M leupeptin. The two forms of cathepsin B which degraded Z-Phe-Arg-NMec, Z-Arg-Arg-NNap and BANA were very sensitive to p-CMB and leupeptin. In addition to cathepsins B and H, a typical cathepsin-L- like activity was found in this fraction but only as a very minor component. The high Mr fraction (Mr = 55 000) contained a cysteine proteinase hydrolyzing, at pH 6.0, Z-Phe-Arg-NMec, and to a lesser extent Z-Arg-Arg-NNap and BANA. Unlike cathepsins B and H, it was very sensitive to p-CMB and HgCl2 and was fully activated only in the presence of 10 mM DTT, and inhibited to 93% by 2.10(-8) M leupeptin. By chromatofocusing, it was resolved into several isoenzymatic forms, eluted between pH 5.8 and 4.0.     

Purification and characterization of beta-N-acetylhexosaminidase I2 from human liver.

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.     

Evaluation of the human sperm proacrosin-acrosin system using gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophesis

Proteolytic enzymes in extracts of human sperm have been identified and partially characterized using a technique which incorporates gelatin into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) system. Initially, semen characteristics from four donors were evaluated. Following this, washed sperm were acid extracted and proacrosin and acrosin activities determined spectrophotometrically. Proteinase activity in unactivated sperm extracts was then extracts was then demonstrated using the gelatin-SDS-PAGE system. Three major (Mr approximately equal to 47,000-54,000) and four faint (Mr approximately equal to 34,000-38,000) bands of digestion were observed. Upon activation of sperm extracts it was observed that maximum esterase activity occurred within 7 min of activation while maximum proteinase activity required approximately 15 min. When gels were washed and incubated in the presence of 50 mM benzamidine, no digestion bands were observed. This indicates that all of the digestion bands were due to trypsin-like proteinases. Finally, upon serial dilution of sperm extracts it was found that this SDS-PAGE system is sensitive enough to detect proteinase activity from as few as 30,000 sperm.