Purification of bovine and human acrosin

Acrosin from human spermatozoa was required for studies of immunological interference with fertilization, but not detailed purification scheme was available for the human enzyme. Since human semen samples cannot be obtained cheaply or in large numbers and contain relatively small amounts of acrosin, development of purification procedures was carried out with bovine semen. Bovine acrosin had not previously been fully purified, and over 1 mg of pure acrosin was obtained from 100 mL of bovine semen, by a process of saline and Triton X-100 washes of the spermatozoa, 1 mM HCl extraction, gel filtration, and ion-exchange and affinity chromatography. The bovine acrosin had a molecular weight (MW) of 39 000 and a specific activity of 93 U/mg, measured with 0.5 mM benzoyl arginine ethyl ester. The same extraction procedure could be followed for human acrosin, but better yields were obtained in the purification if the ion-exchange step was omitted. The human acrosin had a MW of 49 000, and traces of a 38 000 MW component were sometimes observed. From 14 human semen samples, containing initially 7-10 U of acrosin activity, about 2.5 U (approximately 20 micrograms of protein) could be obtained in a pure state.     

Two satisfactory methods for purification of human acrosin

Acrosin has been purified from human sperm cells by two alternative procedures which give purer products and in higher yields than could be achieved previously. The products were characterized by their molecular weight, catalytic action, sensitivity to inhibitors, and reaction with a polyclonal anti-acrosin antibody. After acid extraction of the cells, one method involves removal of acrosin inhibitors by vacuum dialysis, followed by affinity chromatography on a soybean trypsin inhibitor (SBTI) column, and therefore requires that the acrosin be in an active form capable of binding to the inhibitor. The other method involves affinity chromatography on a column of a monoclonal anti-acrosin antibody (MAb) and can be used to provide either active or proenzyme forms of acrosin, by choice of extraction conditions and inclusion of appropriate inhibitors. The yield of human acrosin from the SBTI method was 104% and from the MAb column was 75%. It is hoped that these procedures will make the very scarce human acrosin more readily available for further study.     

Boar acrosin, II: Amino acid composition, amino terminal residue and molecular weight estimations by ultracentrifugation.

The molecular weight of boar acrosin in neutral solution was estimated to be 41000 +/- 1000 by high-speed sedimentation equilibrium analysis. This result is in good agreement with the value found earlier[1] by sodium dodecylsulfate polyacrylamide gel electrophoresis. The sedimentation coefficeint of acrosin obtained by active enzyme centrifugation of partly purified preparations is in accordance with the sedimentation coefficient of the pure preparation estimated by conventional sedimentation velocity analysis. The sedimentation coefficient of acrosin is considerably decreased in slightly acidic solution (pH 4), indicating that changes in the tertiary structure occur upon acidification. The amino acid composition of the acrosin preparation homogeneous by electrophoretic and chromatographic criteria and in sedimentation studies was determined. Valine was found as the unique N-terminal amino acid. However, in microheterogeneous forms of acrosin, alanine and methionine were also detected in end group analysis.     

Intracellular proclotting enzyme in limulus (Tachypleus tridentatus) hemocytes: its purification and properties

A proclotting enzyme associated with the hemolymph coagulation system of limulus (Tachypleus tridentatus) was highly purified from the hemocyte lysate. The first step of purification was performed by chromatography of the lysate on a pyrogen-free dextran sulfate-Sepharose CL-6B column, which was essential for separation of the proclotting enzyme from its activator, named factor B. The following steps consisted of column chromatographies on DEAE-Sepharose CL-6B, Sephadex G-150, benzamidine-CH-Sepharose and Sephacryl S-300. Through these procedures, 1.4 mg of the purified material was obtained from 630 ml of the lysate and approximately 300-fold purification was achieved. The preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence and absence of 2-mercaptoethanol. The single-chain proclotting enzyme was a glycoprotein with an apparent molecular weight of 54,000, and no gamma-carboxyglutamic acid was detected. The proclotting enzyme was converted to its active form by purified factor B or by trypsin. The resulting clotting enzyme had a molecular weight of 54,000, consisting of a heavy chain of Mr = 31,000 and a light chain of Mr = 25,000. The serine active site of the clotting enzyme was found in the heavy chain. The chemical analyses of the isolated heavy and light chains indicated that the activation of the proclotting enzyme to its active form by factor B or trypsin is induced by a limited proteolysis, yielding two chains bridged by a disulfide linkage(s).     

Purification and initial characterization of guinea pig testicular acrosin

Guinea pig (GP) acrosin was purified following acid extraction of testicular acetone powder, pH precipitation of the soluble extract, gel filtration on Sephadex G-100, ion-exchange chromatography on SP-Sephadex, and affinity chromatography on Concanavalin A-Sepharose. Final purification was achieved by re-chromatography on Sephadex G-100. Enzymatic activity was detected by following the hydrolysis of N-benzyloxycarbonylarginyl amide of 7-amino-4-trifluoromethylcoumarin at 37 degrees C, pH 8.0, before and after activation. GP testicular acrosin exhibited a molecular weight of 48,000 by gel filtration and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE in gels containing 0.1% gelatin, protease activity was observed to comigrate with the major protein detected by silver staining. The purified GP acrosin showed cross-reactivity with a monospecific polyclonal rabbit antiserum directed against boar sperm acrosin and exhibited reversible pH-dependent activation. The physiochemical characteristics of the purified protein, including the amino acid composition, resemble those reported for acrosins from other species.     

Isolation of rabbit testicular cathepsin D and its role in the activation of proacrosin

Cathepsin D was purified 900-fold with 30% recovery from rabbit testes using pepstatin bound Sepharose affinity chromatography. The enzyme is homogeneous as observed by acrylamide gel electrophoresis. The heat stable enzyme exhibits an apparent molecular weight of 42,000 with identical subunits of 20,000. Purified cathepsin D catalyses the conversion of proacrosin to acrosin.     

A rapid purification procedure for pyruvate kinase from the hyphal fungus Aspergillus nidulans

Pyruvate kinase was purified from the filamentous fungus Aspergillus nidulans with a 45-55% yield. The procedure involved dye-affinity chromatography and fast protein liquid chromatography, resulting in highly active and pure enzyme in milligram quantities within 2 days. The purified enzyme, a tetramer with a subunit molecular weight of 65,000 and an isoelectric point of 4.7, was used to determine the amino acid composition.     

Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer

Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli. The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK. The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells. Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer.     

Characterization of a fibrinogenase from northern copperhead (Agkistrodon contortrix mokasen) venom

One of the fractions obtained by the carboxymethylcellulose ion-exchange chromatography of northern copperhead (Agkistrodon contortrix mokasen) venom prevented the thrombin-induced clotting of fibrinogen by proteolytically degrading the fibrinogen. The active component has been further purified to apparent electrophoretic homogeneity by molecular sieve chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated a molecular weight of 22 900 +/- 600 for the purified enzyme. In addition to its fibrinogenase activity, it catalyzed the hydrolysis of hide power azure and had an intraperitoneal LD50 value in mice of less than 5.1 microgram/g body weight. The enzyme rapidly destroyed fibrinogen's ability to form clots. Electrophoresis of fibrinogen which had been incubated only a few minutes with the fibrinogenase revealed the rapid disappearance of the alpha-chain and the appearance of lower molecular weight fragments. The neutral pH optimum and ethylenediamine-tetraacetic acid (EDTA) and dithiothreitol sensitivity indicated that this enzyme belonged to the class metalloproteinases. Atomic absorption studies have revealed one zinc atom per molecule of protein. The apoenzyme's activity was restored by incubation with ZnCl2.     

Purification and characterization of two types of trypsin-like enzymes from sperm of the ascidian (Prochordata) Halocynthia roretzi. Evidence for the presence of spermosin, a novel acrosin-like enzyme

Two types of trypsin-like proteases, spermosin and acrosin, have been highly purified from spermatozoa of the ascidian (Prochordata) Halocynthia roretzi by a procedure including diethylaminoethylcellulose chromatography, Sephadex G-100 gel filtration, and soybean trypsin inhibitor-immobilized Sepharose 4B chromatography. Each purified preparation was judged to be homogeneous on the basis of chromatographic analysis and sodium dodecyl sulfate-gel electrophoresis. The molecular weights of spermosin and acrosin were estimated to be 27,000 and 32,000-34,000, respectively, by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the former was 6.5, while that of the latter was 5.5. Non-ionic detergents, e.g. Brij 35, showed marked stabilizing effects on the purified enzymes. Both of these enzymes had pH optima between 8.5 and 9.0, and their activities were enhanced by the addition of calcium chloride. The enzymes were inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, leupeptin, antipain, soybean trypsin inhibitor, aprotinin, ovomucoid, valyl-prolyl-arginyl-chloromethane, glycyl-valyl-arginyl-chloromethane, p-aminobenzamidine, benzamidine, zinc chloride, and mercuric chloride. Lima bean trypsin inhibitor and tosyl-lysyl-chloromethane strongly inhibited acrosin, but not spermosin. While the substrate specificity of acrosin was rather broad, that of spermosin was very narrow; the latter enzyme hydrolyzed only t-butyloxycarbonyl-valyl-prolyl-arginine 4-methylcoumaryl-7-amide among 12 peptidyl-arginine (or lysine) 4-methylcoumaryl-7-amides tested. Thus, the ascidian spermatozoa possess at least two proteases, acrosin and spermosin; the former shows the properties closely related to those of mammalian acrosin (EC 3.4.21.10), but the latter is a unique type of acrosin-like enzyme in respect to the substrate specificity and inhibitor susceptibility.     

Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria.

Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase.     

Phosphofructokinase from Ascaris suum. Purification and properties

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.     

Purification and properties of Aerococcus viridans lactate oxidase

Lactate oxidase was purified from cells of Aerococcus viridans by a procedure which utilized ammonium sulfate fractionation, DEAE Sepharose CL-6B chromatography, and Sephadex G-100 chromatography. The final preparation was homogeneous by SDS-polyacrylamide gel electrophoresis. The enzyme appears to be a tetramer with a subunit molecular weight of 44,000 and utilizes FMN as a cofactor. The enzyme was highly specific for L-lactate. D-lactate, glycolate, and D,L-2-hydroxybutyrate were not oxidized by the enzyme but were competitive inhibitors. The enzyme could be irreversibly inactivated by incubation with bromopyruvate. This inactivation appears to involve a covalent modification near the active site of the enzyme; however, the flavin cofactor is not the site of this modification.     

Comparative activation studies with extracted and purified human proacrosin

Proacrosin was purified from acid extracts of human spermatozoa by concanavalin A precipitation and Bio-Gel P-100 chromatography. Two molecular weight forms of proacrosin were obtained, a major one with a Mr of 70,000-71,000 and a minor one with a Mr of 47,000-53,000. In contrast to sperm extracts, the purified forms of proacrosin were free of acrosin inhibitor(s) and nonzymogen acrosin. By modulating pH, ionic strength and temperature, the activation of proacrosin in sperm extracts was compared to only the major form of purified proacrosin, since it seemed to be the source of the lower molecular weight form of proacrosin. In both preparations, proacrosin activation occurred maximally over a broad pH range (7.6-8.8 for purified proacrosin and 7.6-9.6 for extract). Additionally, an ionic strength of 0.1 and above caused a decrease in proacrosin activation in both preparations. Similarly, proacrosin was sensitive to short incubation periods at 45 degrees C and above which caused a decrease in the amount of proacrosin found in both preparations.     

Purification of Epstein-Barr virus DNA polymerase from P3HR-1 cells.

The Epstein-Barr virus DNA polymerase was purified from extracts of P3HR-1 cells treated with n-butyrate for induction of the viral cycle. Sequential chromatography on DNA cellulose, phosphocellulose, and blue Sepharose yielded an enzyme preparation purified more than 1,300-fold. The purified enzyme was distinct from cellular enzymes but resembled the viral DNA polymerase in cells infected with herpes simplex virus type 1 or 2. The active enzyme had an apparent molecular weight of 185,000 as estimated by gel filtration on Sephacryl S-300. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major polypeptide corresponding to a molecular weight of ca. 110,000. This polypeptide correlated with the catalytic function of the purified enzyme, whereas the other, less abundant polypeptides did not. By immunoblotting, the 110,000-molecular-weight polypeptide could be identified as a viral polypeptide. It could not be determined whether the native enzyme was composed of more than one polypeptide.     

Purification and partial characterization of neuraminidase from type III group B streptococci

Extracellular neuraminidase from a type III fresh clinical isolate of a group B streptococcus was purified by a combination of salt fractionation, affinity chromatography of Affi-Gel blue, ion-exchange chromatography on diethylaminoethylcellulose, and gel filtration on Sephacryl S-200. These procedures yielded enzyme which was purified approximately 1,000-fold compared with the enzyme found in the original supernatant fluid. This type III streptococcal neuraminidase had a molecular weight of approximately 125,000 as estimated by filtration on Sephacryl S-200 and approximately 106,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to the majority of other bacterial neuraminidases, the type III group B streptococcal enzyme had no effect on colominic acid or N-acetylneuramin-lactose; however, it was quite active on bovine submaxillary mucin.     

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. I. Isolation and characterization of multiple forms

The condensation of two propionyl-CoA units or a propionyl-CoA with acetyl-CoA is required for the synthesis of 2-methylvalerate or 2-methylbutyrate, respectively, two of the major fermentation products of Ascaris anaerobic muscle metabolism. An enzyme that preferentially catalyzes the condensation of propionyl-CoA rather than acetyl-CoA has been purified from the mitochondria of the parasitic intestinal nematode Ascaris lumbricoides var. suum. The purified enzyme is over 10 times more active with propionyl-CoA than with acetyl-CoA as substrate. It also catalyzes the coenzyme A-dependent hydrolysis of acetoacetyl-CoA at a rate four times higher than the propionyl-CoA condensation reaction. The purified Ascaris condensing enzyme preferentially forms the 2-methyl-branched-chain keto acids rather than the corresponding straight chain compounds. The native molecular weight of the purified enzyme was estimated to be 160,000 by gel filtration chromatography and 158,000 by high pressure liquid chromatography. The enzyme migrated as a single protein band with Mr 40,000 during sodium dodecyl sulfate-polyacrylamide electrophoresis, indicating that the enzyme is composed of four subunits of the same molecular weight. Chromatography on CM-sephadex resulted in the isolation of two separate peaks of activity, designated as A and B. Both A and B had the same molecular weight and subunit composition. However, they differed in their specific activities and isoelectric points. The pIs of condensing enzymes A and B were 7.6 and 8.4, respectively. Propionyl-CoA was the best substrate for the condensation reaction with both enzymes. However, the specific activity of enzyme B for both propionyl-CoA condensation (3.4 mumol/min/mg protein) and acetoacetyl-CoA thiolysis (13.8 mumol/min/mg protein) was 2.4 times higher than that obtained with enzyme A. Similarly, chromatography on phosphocellulose resolved the Ascaris condensing enzyme activity into one minor and two major peaks. All of these components had the same molecular weight and subunit composition, but differed in their specific activities. The two major phosphocellulose peaks cross-reacted immunologically when examined by the Ouchterlony double immunodiffusion technique. In addition, antiserum against the phosphocellulose most active form cross-reacted with forms A and B isolated by chromatography of the enzyme on CM-Sephadex, indicating that all forms were immunochemically related.     

Purification and properties of the bifunctional proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase from Pseudomonas aeruginosa

Proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified from Pseudomonas aeruginosa strain PAO1. Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid. The purification procedure involved DEAE-cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G-200. It resulted, after 40-fold purification with 11% yield, in a homogeneous preparation (greater than 98% pure). The molecular weight of the single subunit was determined as 119,000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242,000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241,000 and 470,000. Manual Edman degradation revealed proline, alanine and aspartic acid as the N-terminal amino acid sequence. Pro/P5C dehydrogenase was highly specific for the L-forms of proline and delta 1-pyrroline-5-carboxylic acid. Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for delta 1-pyrroline-5-carboxylic acid was non-hyperbolic.     

Purification and some properties of cycloinulo-oligosaccharide fructanotransferase from Bacillus circulans OKUMZ 31B

Cycloinulo-oligosaccharide fructanotransferase was purified from the cultured medium of Bacillus circulans OKUMZ 31B, to electrophoretic homogeneity, by anion-exchange column chromatography on DEAE-Toyopearl 650M, hydrophobic column chromatography on Butyl-Toyopearl 650M, gel-filtration column chromatography on Sephacryl S-2000HR and anion-exchenge column chromatography on SuperQ-Toyopearl 650M. The enzyme has a molecular weight of 132 000 and a pI of 4.1. The enzyme was most active at pH 7.5 and 40°C, and was stable at pH 6.0–9.0 and below 40°C. The enzyme catalyses the conversion of inulin into cycloinulohexaose and cycloinuloheptaose in the ratio of ca. 4:1, and a small amount of cycloinulo-octaose. The enzyme has an isoform which may be a proteolyticaly modified species of the CFTase because of its reduced molecular weight, 126 000.     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.