Sodium Dodecyl Sulfate Disc Gel Research Articles

Purification and partial chemical characterization of a glycoprotein with antifertility activity from human seminal plasma.

The preparation of a highly purified antifertility factor from human seminal plasma is described, and some of its biochemical properties have been determined. Purification was achieved by ultracentrifugation of particle-free human seminal plasma, followed by chromatography of the precipitate using carboxymethyl cellulose, concanavalin A, and Sepharose 6B columns. An occasional contaminant was further removed by preparative isoelectric focusing. During the purification procedures, the activity of the fractions was monitored by mixing them with capacitated mouse spermatozoa for 20 min before adding an aliquot to intact mouse oocytes, and determining fertilization after 24 h. The I50 (amount causing a 50% reduction in fertilization as compared to the control) of the final purified factor was 45 micrograms protein. Purity was established by standard and sodium dodecyl sulfate disc gel electrophoresis, isoelectric focusing, high-pressure liquid chromatography, and sedimentation analysis. These methods, as well as Sepharose gel filtration, were also used for the molecular weight estimations; good agreement was obtained between the various techniques. The factor appears to be a glycoprotein with a molecular weight of about 200,000. It consists of two subunits with molecular weights of about 125,000 and 72,000 and s-20,w of 6.2 s and 4.3 s. The factor contains relatively high amounts of aspartic acid and glutamic acid residues as well as leucine and serine, but only small amounts of tryptophan and no methionine was detected. The carbohydrate fraction is particularly rich in galactose and N-acetylgalactosamine but also contains mannose and N-acetylglucosamine and small amounts of fucose and sialic acid.

Isozymes of S-adenosylmethionine synthetase from rat liver: isolation and characterization.

S-Adenosylmethionine synthetase exists in at least two distinct forms, alpha- and beta-forms, in adult liver. The beta-form was purified to homogeneity from the soluble fraction of rat liver with a yield of about 10%. An antiserum directed against the purified beta-form from rat liver was prepared by injecting the purified enzyme into a rabbit. Ouchterlony double diffusion analysis and immunochemical titrations revealed that the isozymes, alpha- and beta-forms, are identical. Thus, the alpha-form was isolated from rat liver as a single protein using immunoaffinity chromatography against the beta-form. The molecular weights of the beta- and alpha-forms were determined to be 48,000 each by sodium dodecyl sulfate disc gel electrophoresis, and about 100,000, and 200,000, respectively, by Sephacryl S-200 gel filtration. These results indicate that the beta-form consisted of two subunits of 48,000 daltons and the alpha-form of four subunits of 48,000 daltons. The sedimentation coefficient was calculated to be 5.5S for the beta-form and 8.0S for the alpha-form.

Prostaglandin-E2 9-ketoreductase from human uterine decidua vera.

Prostaglandin-E2 9-ketoreductase, the enzyme which catalyzes the reaction from prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), has been purified 232-fold from human uterine decidua vera. The molecular mass of the enzyme, as estimated by fast protein liquid chromatography, was 29 kDa. Sodium dodecyl sulfate disc gel electrophoresis of the denatured enzyme revealed a molecular mass of 31 kDa. These data suggest that the enzyme consists of a single polypeptide chain. The rate equation of the enzyme reaction for two substrates was used for the determination of five kinetic constants. The equilibrium constant with respect to PGE2 was 83 microM, the Michaelis constant, Km, for PGE2 was 93 microM. For NADPH, the equilibrium constant was 1.0 microM and Km was 1.6 microM. The maximal velocity for the forward reaction was V1 = 217 pmol/min. The inhibition constants for the analgesic agents indomethacin and fentiazac were Ki = 850 microM and Ki = 450 microM and for the steroid progesterone Ki = 1.5 mM, respectively. Prostaglandin-E2 9-ketoreductase might be responsible for the control of the PGE2/PGF2 alpha ratio in human decidua vera. The enzyme, therefore, might be an important factor in the cascade of events leading to uterine contractions and parturition.

Cytidine deaminase from Escherichia coli B. Purification and enzymatic and molecular properties.

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from Escherichia coli has been purified to homogeneity through a rapid and efficient two-step procedure consisting of anion-exchange chromatography followed by preparative electrophoresis. The final preparation is homogeneous, as judged by a single band obtained by disc gel electrophoresis performed in the absence and presence of denaturing agents. The native protein molecular weight determined by gel filtration is 56 000. Sodium dodecyl sulfate disc gel electrophoresis experiments conducted upon previous incubation of the enzyme with dimethyl suberimidate suggest an oligomeric structure of two identical subunits of 33 000 molecular weight. The absorption spectrum of the protein reveals a maximum at 277 nm and a minimum at 255 nm. The isoelectric point is at pH 4.35. Amino acid analysis indicates an excess of acidic amino acid residues as well as six half-cystine residues. No interchain disulfide groups have been evidenced. According to Cleland's nomenclature, kinetic analysis shows a rapid-equilibrium random Uni-Bi mechanism. Cytidine deaminase is competitively inhibited by various nucleosides. Km values for cytidine, deoxycytidine, and 5-methylcytidine are 1.8 X 10(-4), 0.9 X 10(-4), and 12.5 X 10(-4) M, respectively.

Immunochemical localization of phenylalanine ammonia-lyase and chalcone synthase in anthers

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) from anthers of the garden tulip "Apeldoorn" have been purified to apparent homogeneity as revealed by sodium dodecyl sulfate disc-gel electrophoresis. Phenylalanine ammonia-lyase was either purified by successive chromatography on Sephacryl S 300 Superfine, HA Ultrogel and on diethylaminoethyl Sephacel or by immunoaffinity chromatography in a single step. Purification of CHS was achieved by chromatography on Sephadex G 200 and on HA Ultrogel followed by chromatofocusing. The purified enzymes were used for the immunization of rabbits. The specificity of the antisera against both PAL and CHS was tested by diverse methods. Antisera against PAL and CHS were employed to detect the localization of the enzymes in cross sections of tulip anthers using an indirect immunofluorometric method. The results show that PAL and CHS are located predominantly in the tapetum cells. These observations strengthen the view that the tapetum plays an important role in the regulation of phenylpropanoid metabolism within the loculus of anthers.

Purification and properties of bovine liver seryl-tRNA synthetase.

Seryl-tRNA synthetase was purified 1800-fold from bovine liver extract by ultracentrifugation at 150 000 X g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, gel chromatography on Sephacryl S-300, adsorption chromatography on hydroxyapatite, affinity chromatography on blue-Sepharose and finally on Matrex gel red A. The relative molecular mass, Mr, in the denatured state was estimated as 87 000 by sodium dodecyl sulfate disc gel electrophoresis; in the active state the Mr, was estimated as 170 000 for the dimeric native enzyme (alpha 2 type) by chromatography on Sephacryl S-300. The amino acid composition of the enzyme was determined. The Km values for ATP and serine were 0.49 mM and 30 microM, respectively. The Km values for tRNASerIGA and tRNASerCmCA were 1.40 microM and 1.25 microM, respectively. Sequences common to the two isoaccepting tRNASer molecules are discussed in relation to the recognition mechanism of the purified seryl-tRNA synthetase.

Inhibition of human activated Factor X by antithrombin III and alpha 1-proteinase inhibitor in human plasma.

The inhibition of activated human Factor X by human plasma protease inhibitors was investigated in both purified and plasma systems. In the former, antithrombin III, alpha 1-proteinase inhibitor, and alpha 2-macroglobulin, at normal plasma concentrations, markedly inhibited activated Factor X. Significant inhibition by alpha 2-antiplasmin, however, was only achieved when present at 40 times its plasma concentration. The relative rates of inhibition of activated Factor X coagulant activity in normal human plasma, antithrombin III-deficient plasma, and alpha 1-proteinase inhibitor-deficient plasma were 1.0, 0.63, and 0.75, respectively. From these relative rates of inhibition and their measured concentrations in the 3 plasmas, it was calculated that antithrombin III, alpha 1-proteinase inhibitor, and alpha 2-macroglobulin contribute 53, 35, and 12%, respectively, to the inhibition of activated Factor X in normal human plasma. Using 125I-labeled activated Factor X and a combination of sodium dodecyl sulfate disc gel electrophoresis and immunoadsorption, it was then demonstrated that antithrombin III accounted for 45-55%, alpha 1-proteinase inhibitor, 35-40%, and alpha 2-macroglobulin, 10-15% of the inhibition of the labeled protease in normal human plasma. The values obtained with the deficient plasmas are consistent with the distribution of activated Factor X-inhibitor complexes in normal human plasma. These data show by two independent techniques, one measuring coagulant activity and the other 125I-labeled activated Factor X-inhibitor complexes, that both antithrombin III and alpha 1-proteinase inhibitor are the major inhibitors of activated Factor X in normal human plasma.

Isolation and characterization of rabbit skeletal muscle protein phosphatases C-I and C-II.

Previous studies have shown that phosphorylase phosphatase can be isolated from rabbit liver and bovine heart as a form of Mr approximately 35,000 after an ethanol treatment of tissue extracts. This enzyme form was designated as protein phosphatase C. In the present study, reproducible methods for the isolation of two forms of protein phosphatase C from rabbit skeletal muscle to apparent homogeneity are described. Protein phosphatase C-I was obtained in yields of up to 20%, with specific activities toward phosphorylase a of 8,000-16,000 units/mg of protein. This enzyme represents the major phosphorylase phosphatase activity present in the ethanol-treated muscle extracts. The second enzyme, protein phosphatase C-II, had a much lower specific activity toward phosphorylase a (250-900 units/mg). Phosphatase C-I and phosphatase C-II had Mr = 32,000 and 33,500, respectively, as determined by sodium dodecyl sulfate disc gel electrophoresis. The two enzymes displayed distinct enzymatic properties. Phosphatase C-II was associated with a more active alkaline phosphatase activity toward p-nitrophenyl phosphate than was phosphatase C-I. Phosphatase C-II activities were activated by Mn2+, whereas phosphatase C-I was inhibited. Phosphatase C-I was inhibited by rabbit skeletal muscle inhibitor 2 while phosphatase C-II was not inhibited. Both enzymes dephosphorylated glycogen synthase and phosphorylase kinase, but displayed different specificities toward the alpha- and beta-subunit phosphates of phosphorylase kinase (Ganapathi, M. K., Silberman, S. R., Paris, H., and Lee, E. Y. C. (1980) J. Biol. Chem. 246, 3213-3217). The amino acid compositions of the two proteins were similar. Peptide mapping of the two proteins showed that they are distinct proteins and do not have a precursor-proteolytic product relationship.

Inactivation of yeast fructose-1,6-bisphosphatase. In vivo phosphorylation of the enzyme.

Incorporation of 32P into yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) was observed after addition of glucose to a cell suspension incubated with (32P)orthophosphoric acid. The 32P counts were coincident with the enzyme band when immunoprecipitates were subjected to sodium dodecyl sulfate disc gel electrophoresis. The incorporation of phosphate was associated with a decrease in enzyme activity. Approximately 1 mol of phosphate was incorporated/mol of enzyme. The phosphate is bound to the enzyme in a phosphoester linkage with a serine residue. Release of 32P accompanying enzyme reactivation was observed both in vivo and in cell-free extracts.

Effects of Light and of Fusarium solani on Synthesis and Activity of Phenylalanine Ammonia-Lyase in Peas

Phenylalanine ammonia-lyase was purified from peas, and a specific antiserum against the enzyme was produced in rabbits. The antiserum was used to study the first 8 hours of the phenylalanine ammonia-lyase activity response in two different organs of the pea from different developmental stages and in response to two different stimuli. Etiolated seedlings were pulse-labeled with l-[(35)S]methionine after either no light exposure or after specific periods of irradiation with blue light. Immature pods were pulse labeled with mixed l-[(3)H]amino acids after specific time periods following inoculation of the pod endocarp surfaces with macroconidia of Fusarium solani. Immunoprecipitates isolated from extracts of each group were analyzed with sodium dodecyl sulfate disc gel electrophoresis and were found to contain a radioactive protein with an electrophoretic mobility identical to that of the phenylalanine ammonia-lyase subunit (M(r) 81,000). The radioactivity contained in the subunit band was interpreted as being due to de novo synthesis of the enzyme. The net rate of phenylalanine ammonia-lyase labeling, found to be initially low in both tissue types, rose dramatically, peaking at approximately a six- to ten-fold greater level at 4 hours after the beginning of the stimulus. Thereafter, the rate of labeling declined slowly. Inoculation with F. solani f. sp. pisi, a true pathogen of peas, caused a fifty per cent greater rate of peak labeling than did inoculation with a nonpathogen, F. solani f. sp. phaseoli. The time profile of the changing rate of labeling correlates with the changing activity level of the enzyme which peaks at 12 hours after the onset of the stimulus. The data presented favor a model which explains the changing activity of phenylalanine ammonia-lyase as being due to a changing rate of synthesis or degradation (or both) of the enzyme rather than due to the activation of a preformed zymogen.

Isolation and characterization of a mitogenic substance for murine and human B lymphocytes from Ulex europeus seeds.

A mitogenic substance, which stimulates murine and human B lymphocytes, has been isolated from the seeds of Ulex europeus. The m.w. of this mitogen was estimated to be approximately 100,000 by sodium dodecyl sulfate disc gel electrophoresis. Under physiologic conditions Ulex mitogen was in the form of a polymer. The isolated mitogen was found to contain 80% carbohydrate, in which glucose, galactose, and rhamnose were the predominant sugars. The carbohydrate portion of the mitogen could be responsible for the mitogenic activity, since the activity was destroyed by periodate treatment but not by protease digestion. It is unlikely that the mitogenic activity is due to the contamination by lipopolysaccharide (LPS), because Ulex mitogen strongly activated spleen cells from C3H/HeJ mice and did not contain a detectable amount of fatty acid. Spleen cells from nude mice, T-depleted spleen cells from conventional mice, or T-depleted spleen cells from conventional mice, or T-depleted lymphocytes from human peripheral blood responded as strongly as lymphocytes (T + B) from normal origin as measured by 3H-thymidine incorporation. It seems that the mitogenic effect is primarily on B cells. Ulex mitogen also induced the increase of immunoglobulin synthesis in murine and human B cells.

Comparison of cartilage structural glycoproteins with matrix proteins and fibronectin

After extraction of proteoglycans and soluble matrix proteins from canine puppy rib cartilage, with 4 M guanidine . HCl, the insoluble collagen-containing residue has been shown to contain two collagenase-resistant structural glycoproteins, A and G. The characteristic subunits of these insoluble structural glycoproteins have been identified by solubilizing them with 50 mM dithiothreitol (DTT) in 1% sodium dodecyl sulfate (SDS) and 8 M urea and comparing the SDS disc gel patterns with those of more readily soluble matrix proteins. Three subunit bands which did not occur in gels from the soluble matrix proteins were found in the solubilized material from both the collagen-containing residue and the structural glycoproteins. One band, of about 87 000 daltons, was found equally in both A and G glycoproteins. The other two bands formed a closely spaced doublet, of about 30 000 and 27 500 daltons, of which the lower band is present in higher concentration in G. Although none of these SDS gel bands corresponds with the 220 000 dalton band produced by pure fibronectin under the same conditions, a fraction of the solubilized glycoprotein material resembles fibronectin in showing an affinity for collagen, fibrinogen, heparin, and an antibody to plasma fibronectin. Crude fibronectin from human plasma contains minor components (including one of about 87 000 daltons) which show partial identify in immunodiffusion reactions with components of the solubilized cartilage structural glycoproteins. Solubilized A gave a stronger reaction with anti-plasma fibronectin than did G and the soluble matrix proteins have no reaction. It is possible either that the intercellular structural glycoproteins are formed by selection of some of the partial cleavage products of fibronectin which occur in connective tissues as well as in plasma, or that cleavage products of tissue structural glycoproteins occur in plasma which cross-react with anti-plasma fibronectin.

Human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase. Two activities at a single enzyme active site.

Two soluble enzyme activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, present in the cytosol fraction of term human placenta, were co-purified with a constant ratio of specific activities, approximating 100:1, respectively. The "pure enzyme" is a single band on sodium dodecyl sulfate disc gel electrophoresis. To evaluate whether catalysis of the estrogen and progestin substrates occurs at a single active site, alkylation studies using 16 alpha-bromoacetoxyprogesterone were designed. This affinity alkylating steroid binds at the enzyme-active site (km 256 microM; Vmax = 0.012 mumol/min/mg), inactivates the enzyme in an irreversible and time-dependent manner which follows pseudo-first order kinetics, and causes coincident loss of both the 17 beta- and 20 alpha-activities. Affinity radioalkylation studies using 16 alpha-[2'-3H]bromoacetoxyprogesterone indicate that 2 mol of steroid bind per mol of inactivated enzyme dimer (Mr = 68,000). Amino acid analyses of the acid hydrolysate of radioalkylated enzyme show that 16 alpha-bromoacetoxyprogesterone dicarboxymethylates a histidyl residue in the active site. These results are identical with those reported for 16 alpha-[2'-3H]bromoacetoxyestradiol 3-methyl ether inactivation and radioalkylation of identically purified "17 beta-estradiol dehydrogenase." Computer graphics were used to construct a model in which: 1) binding of estrogen and progestin substrates at one active site permits stereospecific catalysis; 2) the estrogen and progestin analogs' alkylating side arms have access to a common histidine residue. These observations clearly demonstrate that the catalysis of estrogen and progestin substrates can occur at a single active site of one enzyme.

Recombinant hormones from fragments of human growth hormone and human placental lactogen.

Peptide fragments generated by limited plasmin digestion and reduction of human growth hormone (hGH) and human placental lactogen (hPL) were recombined to produce biologically active molecules consisting of the NH2-terminal two-thirds of one hormone molecule linked to the COOH-terminal one-third of the other. Plasmin-cleaved hPL and hGH each consist of an NH2-terminal fragment (1-134) connected to a COOH-terminal fragment (141-191) through a Cys53-Cys165 disulfide bond. The plasmin-cleaved hormones were reduced, the fragments isolated, and then recombined in equimolar quantities to prepared recombinant hormones. On sodium dodecyl sulfate disc gel electrophoresis the recombinants migrated as a single band in the position of native hormone when they were not reduced and as two bands in the positions of the fragments when they were reduced. This indicated that the recombinants had re-formed a disulfide bond. The derivatives were tested for immunologic activity in hPL and hGH radioimmunoassays, for lactogenic and growth-promoting activity by the radioreceptor assays using membranes from mammary gland and liver of lactating rabbits, and for secondary and tertiary structure by circular dichroism measurements in the near and far UV regions. The recombinant with NH2-terminal hGH had hGH immunologic activity, hGH structure and lactogenic and growth-promoting activity, while the hybrid with NH2-terminal hPL had hPL immunologic activity, hPL structure, and lactogenic activity alone. Therefore the immunologic activity, biologic activity, and structure of each of the covalently linked derivatives was characteristic of the hormone's NH2-terminal 1-134 fragment. The COOH-terminal 141-191 sequence maintains overall conformation but appears to have little, if any, role in determining biologic specificity.

Purification and properties of dihydrothymine dehydrogenase from rat liver.

Rat liver dihydrothymine dehydrogenase, the rate-limiting enzyme of thymidine and uridine degradation, was purified to homogeneity as judged by polyacrylamide disc gel electrophoresis, sedimentation velocity, and Ultrogel ACA-34 elution profile. The enzyme has a molecular weight of 220,000 +/- 5,000 as determined by Ultrogel ACA-34 and sedimentation equilibrium The s20,w value of the enzyme was 9.2 S. The isoelectric point was at pH 5.25. The enzyme is composed of two identical subunits of an approximate molecular weight of 110,000 +/- 3,000 as determined by sodium dodecyl sulfate disc gel electrophoresis. The enzyme contains 4 mol of FAD and 3 mol of iron per mol of enzyme. Flavin released from the enzyme by boiling was identified as FAD by absorption spectra and thin layer chromatography, indicating that the enzyme is a flavometal protein. During dialysis, the enzyme was stabilized by 2-mercaptoethanol, but neither NADPH nor thymine was effective. The relative rates of reduction of pyrimidine analogues substituted at position 5 were 5-fluorouracil > 5-bromouracil > 5-diazouracil > 5-iodouracil > 5-nitrouracil, with 5-fluorouracil and 5-diazouracil 70% faster than thymine. Uracil was reduced 25% faster than thymine. The pH optimum for the forward and reverse reactions was 7.4. In the presence of NADPH, the apparent Km was 2.6 microM for thymine and 1.8 microM for uracil. Apparent Km for NADPH was 15 microM with thymine as substrate and 11 microM with uracil. In the reverse reaction, apparent Km values were 43 microM for dihydrothymine and 193 microM for dihydrouracil; apparent Km for NADP+ was 3.8 microM with dihydrothymine as substrate and 2.9 microM with dihydrouracil.

Purification and characterization of a ferredoxin from Rhizobium japonicum bacteroids.

An eight-iron, eight-sulfur ferredoxin from Rhizobium japonicum bacteroids of soybean root nodules has been purified to apparent homogeneity as judged by disc gel electrophoresis. The purification procedure included chromatography on DEAE-cellulose, Bio-Gel P-60, and hydroxylapatite. Specific activities of several purified preparations of bacteroid ferredoxin ranged from 1700 to 1900 nmol of C2H4 produced . min-1 . mg-1 in the reaction mediating electron transfer between illuminated chloroplasts and bacteroid nitrogenase. A molecular weight of 6740 for the protein was determined by low speed sedimentation equilibrium and a molecular weight of 6500 was estimated from the mobility of bacteroid ferredoxin relative to the mobility of standard proteins during sodium dodecyl sulfate disc gel electrophoresis. All of the common amino acids were present except arginine, methionine, and tryptophan. The absorbance spectrum of the oxidized protein exhibited maxima at 285 nm and 380 nm with a shoulder near 305 nm. The A380/A285 ratio was 0.76 and the extinction coefficient at 380 nm for the oxidized protein was found to be 30,800 M-1. Equilibration of bacteroid ferredoxin with methyl viologen at various potentials revealed a midpoint oxidation-reduction potential of -484 mV. Spectrophotometric examination of iron-sulfur clusters extruded from bacteroid ferredoxin with benzenethiol and the transfer of its iron-sulfur clusters to other ferredoxins established the presence of two [4Fe-4S] clusters in a molecule of bacteroid ferredoxin. The EPR spectrum of oxidized ferredoxin consisted of a small signal at g = 2.02 integrating to 0.19 spin/molecule. The EPR spectrum of ferredoxin reduced with 5-deazaflavin exhibited a signal with features at g values of 1.88, 1.94, 2.01, and 2.07, and integrated to 1.7 spins/molecule. The EPR properties of bacteroid ferredoxin are characteristic of a ferredoxin operating between the 1+ and 2+ oxidation levels. Bacteroid ferredoxin mediated electron transfer to clostridial hydrogenase, but was not reduced by the clostridial phosphoroclastic system in the presence of pyruvate. Bacteroid ferredoxin reduced by illuminated 5-deazariboflavin also supported a high rate of C2H2 reduction by bacteroid nitrogenase which was free of Na2S2O4. It was concluded, on this basis, that bacteroid ferredoxin has the capability of functioning as the electron donor for nitrogenase in R. japonicum.

Purification and characterization of fatty acid cyclooxygenase from human platelets.

The fatty acid cyclooxygenase (EC 1.14.99.1) that produces the prostaglandin, thromboxane, and prostacyclin precursor (PGH2), was solubilized from human platelet microsomes in 20 sucrose and 1.0% Triton X-100. The enzyme was purified 300-fold by electrofocusing, Sephadex G-200 gel filtration, and hydrophobic chromatography on ethyl agarose. The cyclooxygenase catalyzed the conversion of arachidonic acid to prostaglandin endoperioxide, PGH2, that was trapped at -25 degrees C and separated on TLC at -20 degrees C. PGH2 was hydrolyzed to HHT in acidic pH, or was chemically converted to PGE2 in slightly alkaline pH in the absence of cofactors. The enzyme showed a broad pH optimum in the range of 7-9. Hemin containing substances such as methemoglobin were absolutely required as cofactors, while tryptophan, epinephrine, phenol, and hydroquinone stimulated the PGH2 formation. Metal ions, such as ZN2+ and Cd2+ inhibited the enzyme reaction at 0.1 to 1 mM. The molecular weight of the purified enzyme was estimated at 79,432 by sodium dodecyl sulfate disc gel electrophoresis at pH 8.0. The properties of the human platelet enzyme was generally similar to the sheep vesicular enzyme in the method of solubilization, pH optimum, and molecular weight.

Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques

1. 1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate containing lysophospholipase II in enzymically active form could be isolated. 3. 3. Microsomal lysophospholipase activity was completely inhibited by [ 3H]-diisopropylphosphofluoridate. Enzyme labelled in this way was isolated by immunoprecipitation from control and chymotrypsin-treated microsomes. Sodium dodecyl sulfate disc gel electrophoresis of the immunoprecipitates showed that chymotrypsin treatment of intact microsomes had no influence on the molecular weight of the enzyme. 4. 4. Attempts to label the lysophospholipase II in microsomes by lactoperoxidase catalyzed iodination or by reaction with the diazonium salt of [ 125I]iodo-sulfanilic acid were negative, although both techniques labelled other microsomal proteins efficiently. 5. 5. Antibody absorption experiments gave no indication for the presence of lysophospholipase antigenic sites on the outside surface of microsomes. 6. 6. These experiments are interpreted to indicate that lysophospholipase II is exclusively located at the luminal side of the microsomal membrane.

Fungal glycosidases. V. Purification and properties of an extracellular exo-(1 leads to 3)-beta-D-glucosidase from Trichophyton mentagrophytes.

An exo-(1→3)-β-D-glucosidase was isolated from the culture filtrate of the fungus Trichophyton mentagrophytes by ammonium sulfate fractionation, gel chromatography on Sephadex G-200 equilibrated with 0.1M sodium phosphate buffer (pH 7.5), and ion exchange chromatography on DEAE-Sephadex A-50. The enzyme was purified 304 fold, with a recovery of 15%. The Km value of the enzyme with pnitrophenyl-β-D-glucopyranoside was 3.1 mM. The enzyme was strongly inhibited by Ag+, Hg2+, Cu2+ and Co2+ ions and also by N-bromosuccinimide and I2. This enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside but did not show any activity towards p-nitrophenyl-α-D-glucopyranoside or other p-nitrophenyl-glycosides. The enzymes also hydrolyzed (1→3)-β-linked glucosides, but not (1→4)-β-and (1→6)-β-linked glucosides. When the enzyme hydrolyzed (1→3)-β-linked oligo and polyglucosides, the only product identified by paper chromatography was glucose monomer. The molecular weight of the enzyme, as determined by sodium dodecyl sulfate disc gel electrophoresis, was 4.9×104 daltons.

Purification and properties of guinea pig antithrombin III.

Guinea pig antithrombin III has been purified from plasma by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose chromatography, isoelectric focussing, and Sephadex G-100 gel filtration chromatography. The final product was homogeneous as judged by sodium dodecyl sulfate disc gel electrophoresis. Purification was 202-fold with a yield of 41%. Antiproteinase activity of antithrombin III was determined by progressive inactivation of thrombin coagulant and amidolytic activity. Heparin cofactor activity was demonstrated by immediate inactivation of thrombin by antithrombin III in the presence of minute quantities of heparin. It also could be demonstrated that thrombin inactivation by antithrombin III occurs by formation of a bimolecular complex whose rate of formation is markedly enhanced by minute quantities of heparin.