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PubMed1977

Purification and properties of the pyrrolidonecarboxylate peptidase of Streptococcus faecium.

Pyrrolidonecarboxylate peptidase (EC 3.4.11.8) from Streptococcus faecium was purified by fractionation with streptomycin sulphate and ammonium sulphate, by chromatography on Sephadex G200 and DEAE-cellulose, and by preparative electrophoresis on Sephadex G25. The purified enzyme on acrylamide gel showed a strong protein band which contained enzyme activity and a very faint band which had no activity. The subunit molecular weight of the purified enzyme was estimated by acrylamide gel electrophoresis in sodium dodecyl sulphate to be 42,000 +/- 1,000. The enzyme showed optimum activity at pH 7.6 and was unstable in the absence of 2-mercaptoethanol. The sensitivity of the enzyme to alkylating agents (N-ethylmaleimide and iodoacetamide) suggested that free sulphydryl groups were essential for enzyme activity. The enzyme was rapidly inactivated above 45 degrees C. The values of the Michaelis constants (Km) obtained with various L-pyrrolidonecarboxylyl dipeptides were similar although there was a 10-fold range in the maximal rates of hydrolysis of these substrates. Inhibition studies showed that the substrate analogues 2-pyrrolidone and pyrrolidonecarboxylate are competitive inhibitors of the enzyme. The binding of substrates and inhibitors to the active site of the enzyme is discussed.

Aminopeptidases↗

PubMed1977

9S binding protein for androgens and progesterone.

A steroid binding protein fraction with a sedimentation coefficient of approximately 9 S (molecular weight approximately equal to 200,000) has been identified in 105,000 X g supernatants of several androgen-responsive organs. Highest concentrations were found in epididymis and testis, but small amounts were detected in prostate, seminal vesicle, kidney, submandibular gland, and lung. The 9S protein binds [3H]dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and [3H]progesterone (4-pregnene-3,20-dione) with equilibrium binding constants of approximately 10(5) M-1 and 10(6) M-1, respectively. The concentration of 9S binding sites in epididymis is approximately 10(-11) mol/mg of supernatant protein, which is at least 10(5) times greater than the concentration of androgen receptor. 9S binding protein appears to be a nonsecretory, intracellular protein and has properties different from the andorgen receptor. It is unretarded on DEAE-Sephadex chromatography at pH 8.0, and its sedimentation rate on sucrose gradients is not altered at high ionic strength (0.4 M KCl). Like the androgen receptor, its binding activity, which is maximal between pH 7 and 9.5, is heat labile, decreased by sulfhydryl reagents, and enhanced by 2-mercaptoethanol. It is suggested that because of its high concentration and low affinity, 9S binding protein may function in the intracellular accumulation of compartmentalization of androgens or progesterone.

Androstane-3,17-diol↗

PubMed1977

Stimulation of human platelet guanylate cyclase by unsaturated fatty acid peroxides.

Guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity of human platelet homogenates was stimulated by the addition of phospholipase A2 or unsaturated fatty acids such as oleic, vaccenic, linoleic, linolenic, eicosenoic, eicosadienoic, and arachidonic acids. The addition of lipoxidase potentiated the fatty acid-induced stimulation of guanylate cyclase purified by DEAE-cellulose column chromatography. The extent of the stimulation was dependent on the concentration of the oxidized form of these fatty acids (peroxides). Saturated fatty acids such as stearic and arachidic acids had no effect on the guanylate cyclase activity in the presence or absence of lipoxidase, indicating that human plateletguanylate cyclase is stimulated by unsaturated fatty acid peroxides rather than by fatty acids. Hemoglobin prevented the enzyme stimulation produced by low concentrations of fatty acid peroxides, but enhanced stimulation of the enzyme activity with high concentrations of fatty acid peroxides. 2-Mercaptoethanol, dithiothreitol, and N-ethylmaleimide inhibited the guanylate cyclase activities both in the presence and absence of unsaturated fatty acidperoxide. The stimulation of guanylate cyclase activity by unsaturated fatty acid peroxidesis attributed to oxidation of sulfhydryl residues of the enzyme protein.

Arachidonic Acids↗

PubMed1976

A clottable protein (coagulogen) from amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus). Its isolation and biochemical properties.

A clottable protein, named coagulogen, was highly purified from the amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus) by a method similar to that used for the lysate of Limulus polyphemus amoebocytes. The isolated material gave a single protein band on analytical gel electrophoresis at pH 3.2, gel electrofocusing, and sodium dodecyl sulfate (SDS) gel electrophoresis with or without 2-mercaptoethanol. It was 90 percent coagulable, and the total yield from 10 ml of the amoebocyte lysate was about 40 mg. The sedimentation coefficient of purified coagulogen was 2.6 S and its molecular weight was estimated to be about 15,300 by sedimentation equilibrium analysis. The molecular weight estimated by SDS-gel electrophoretic analysis was 19,500 +/- 1,000. This discrepancy was apparently due to abnormal mobility arising from the basic nature of this protein on electrophoresis. The protein had a high isoelectric point of pH 10.0 +/- 0.2, as measured by the isoelectric focusing technique. It consisted of a total of 132 to 135 amino acid residues and contained high levels of basic amino acids, which accounted for more than 16 per cent of the total amino acid residues. No methionine was detected. High contents of valine, half-cystine, glutamic acid (glutamine), and phenylalanine were found. The N-terminal sequence of the first three residues of the coagulogen was Ala-Asx-Thr, and its C-terminal residues was identified as phenylalanine, indicating that it consists of a single polypeptide chain. It is of interest that the first three N-terminal residues are homologous with those of the Aalpha-chain of non-human primate fibrinogen.

Amino Acids↗

PubMed1977

Purification and characterization of guinea-pig epidermal acid phosphatase.

Guinea-pig epidermal acid phosphatase has been purified approximately 120-fold by a procedure including acid treatment, CM-cellulose and DEAE-cellulose chromatography, and gel filtration on Sephadex G-100. The enzyme had a pH optimum at 5-0 and the optimal temperature for activity was approximately 50 degrees C. The enzyme was not activated by divalent cations or 2-mercaptoethanol, but it was inhibited by p-chloromercuribenzoate and by fluoride. The km value for p-nitrophenyl phosphate was 1-31x10-4 M, the molecular weight was about 73,000 as determined by Sephadex G-100 gel filtration and the isoelectric point was 6.1. The enzyme hydrolyzed deoxyribonucleoside monophosphates to deoxyribonucleosides.

Acid Phosphatase↗

PubMed1976

Properties of Fcgamma receptors in normal and malignant human tissues.

Properties of the IgG receptors were studied by treating tissue sections or suspensions of peripheral mononuclear cells with various chemical reagents and determining changes in their ability to react with IgG-sensitized erythrocytes (EA). A heterogeneity of the Fc receptors was revealed. Iodoacetamide, 2-mercaptoethanol, sodium azide, EDTA, and a pH varying from 6.0 to 8.2 had no effect on Fc receptors in sections of normal lymphoreticular tissue and malignant tissue. Formaldehyde, low pH, and high salt concentrations affected receptor activity to various degrees. Receptors in liver sections and on monocytes were generally more resistant than receptors in spleen sections and on B lymphocytes. Receptors in malignant tissue behaved either like receptors in spleen or like receptors in liver. Although all tissues were sensitive to periodic acid, the Fc receptors in malignant tissue were always more resistant.

Azides↗

PubMed1975

Hydroxylation of p-Coumaric acid by illuminated chloroplasts. The role of superoxide.

1. Chloroplasts isolated from leaves of spinach-beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p-coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction. 2. Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect. 3. Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2-mercaptoethanol. 4. It is proposed that O-2- generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.

Ascorbic Acid↗

PubMed1976

Fluroescence of proteins in 6-M guanidine hydrochloride. A method for the quantitative determination of tryptophan.

To determine the tryptophan content in proteins,an analytical ultraviolet fluroescence method is proposed based on making uniform the environment of aromatic chromophores in 6-7 M guanidine hydrochloride. The fluorescence intensity scale is calibrated using standard solutions of free tryptophan. A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins. This method is particularly suited to proteins carrying groups absorbing in the 290-370 nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2-mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods. It is less time-consuming than techniques requiring hydrolysis or chemical reactions.

Guanidines↗

PubMed1976

Membrane-bound DD-carboxypeptidase and transpeptidase activities from Bacillus megaterium KM at pH 7. General properties, substrate specificity and inhibition by beta-lactam antibiotics.

1. The membranes from Bacillus megaterium KM contained a DD-carboxypeptidase with optimum activity under the following conditions: pH 7; ionic strength, 1.3 M; temperature, 40 degrees C and below 20 degrees C. It did not require any divalent cation, but was inactivated by Cu2+ and Hg2+. It was stimulated by 2-mercaptoethanol and low concentrations of p-chloromercuribenzoate. 2. The membrane preparation also catalyzed a simple transpeptidation reaction using as carboxyl acceptors D-alanine or glycine. 3. The conditions for optimum activity, temperature-inactivation, temperature-dependence of the activity, carboxyl donor specificity, sensitivity to beta-lactam antibiotics, and insensitivity to potential peptide inhibitors of both enzyme activities, was identical. The DD-carboxypeptidase showed inhibition by D-alanine and Ac2-L-Lys-D-Ala. 4. The inhibition by beta-lactam antibiotic was reversible for both enzymic activities and the time-dependence for their recovery was identical. 5. The DD-carboxypeptidase was very sensitive to changes in the configuration and size of the side-chains of the C-terminal dipeptide of the substrate. Amino acid residues at the C-terminus that precluded the peptide from being a DD-carboxypeptidase substrate were not acceptors in the transpeptidation reaction. Dipeptides were not acceptors for the 'model transpeptidase'. 6. It is suggested that both activities are catalysed by the same enzyme molecule, whose physiological role is not the formation of peptide crosslinks during peptidoglycan biosynthesis.

Acyltransferases↗

PubMed1977

A deoxyribonuclease from Chlamydomonas reinhardii. 1. Purification and properties.

A deoxyribonuclease has been purified more than 2000-fold from the green algae, Chlamydomonas reinhardii. The enzyme is most active on denatured DNA. Optimum activity is at pH 8.5, in 80 mM Tris-HCl buffer and 2 mM CaCl2. Other divalent cations can replace Ca2+ with varying lower efficiency. EDTA and inorganic phosphate are strongly inhibitory, while ATP and high concentrations of 2-mercaptoethanol are slightly inhibitory. The molecular weight is approximately 35 000, the Stokes radius is 2.7 nm, and the sedimentation coefficient 2.8 S. It is a single polypeptide chain, and the frictional ratio of 1.27 suggests it is only slightly asymetrical. The isoelectric point is 9.5. This enzyme has been termed exonuclease 1.

Adenosine Triphosphate↗

PubMed1977

Complement-fixing antibody response in pneumococcal pneumonia.

Previous studies of complement-fixing antibodies to pneumococcal capsular polysaccharides in humans have yielded conflicting results. We studied 65 sera from 25 patients with pneumococcal pneumonia, using both fresh sera and heat-inactivated sera with added human complement. Only 4 of the 25 patients developed detectable levels of complement-fixing anticapsular antibody. Of the 25 patients, 22 developed detectable levels of hemagglutinating anticapsular antibody, indicating that they were able to develop an immunological response during the infection. Most of the antibody detected by hemagglutination was sensitive to 2-mercaptoethanol, but some 2-mercaptoethanol-resistant antibody was also detected. In studies with rabbit antiserum, the complement fixation test was found to be as sensitive as the hemagglutination test for detection of anticapsular antibody. It is not clear why detectable levels of complement-fixing antibody do not develop more often in patients with pneumococcal pneumonia. Studies of purified anticapsular antibody would be of interest to determine whether or not these antibodies are restricted to immunoglobulin subclasses having a limited capacity to fix complement.

Adult↗

PubMed1977

Multiple leukocyte abnormalities in chronic granulomatous disease: a familial study.

A variety of leukocyte enzyme activities were studied in an 11-year-old female with chronic granulomatous disease (CGD) and several members of her family. Leukocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity was 17 nmol/min/mg protein in the patient; two brothers with symptoms of recurrent bacterial infections have G-6-PD activities of 58 and 37 nmol/min/mg protein; the activites of this enzyme in both parents, maternal grandmother, and one additional brother were within normal limits. Storage at 4 degrees or heating at 37 degrees over a 120-min period revealed a marked lability of G-6-PD activity in the patient's cells which could not be stabilized by the addition of NADP and 2-mercaptoethanol; this lability was not seen in other family members tested. Activities of leukocyte glutathione reductase were reduced in both parents and the two affected male siblings with values of 18, 23, 23, and 24 nmol/min/mg protein, respectively. Activities of leukocyte glutathione peroxidase were reduced in all of the immediate family members tested, with values ranging from 11.2 to 43 nmol/min/mg protein; the activity of this enzyme in the patient was 38.5. Leukocyte NADP content in the patient, father, and two affected male siblings were 16.5, 23.4, 22.2, and 28.2 nmol/15 min/10(7) leukocytes, respectively.

Adolescent↗

PubMed1976

Purification and characterization of N-hydroxy-2-acetylaminofluorene sulfotransferase from rat liver.

N-Hydroxy-2-acetylaminofluorene (N-OH-2-AAF) sulfotransferase is an enzyme that catalyzes the sulfate transfer from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to N-OH-2-AAF to form a highly reactive product acetylaminofluorene N-sulfate. It has been purified about 2000-fold with a yield of over 12% from adult Sprague-Dawley male rat livers by an eight-step procedure. The final preparation was homogeneous on analytrical disc gel electrophoresis. The purified enzyme had activity toward p-nitrophenol with an approximately 1600-fold increase in specific activity over the crude homogenate, but it had almost no detectable activity toward steroids such as estrone, beta-estradiol, testosterone, dehydroisoandrosterone, and corticosterone. There was also very little sulfation activity toward serotonin and L-tyrosine methyl ester. The optimal pH for the enzyme activity is approximately 6.3 when measured in sodium phosphate buffer. Mg2+ at 6 to 9 mM could increase the enzyme activity up to 30%. Mn2+ activated the enzyme only slightly at very low concentrations. Zn2+, Co2+, Cu2+, and Ni2+ were all strongly inhibitory, but Ca2+ had very little effect. Thiol compounds were found to have a stabilizing effect and thiol-blocking reagents were potent inhibitors for this enzyme. The pure enzyme was very unstable especially in diluet solutions. The isoelectric point (pl) of the enzyme is 5.66 +/- 0.07. The molecular weight of the native enzyme was 68,000 +/- 500 as estimated by Sephadex G-100 and G-200 gel filtrations. A single component with molecular weight of 38,250 +/- 1,350 was observed on sodium dodecyl sulfate gel electrophoresis in the absence and presence of 2-mercaptoethanol. Comparison of the enzyme activity in mail and female rat livers at each stage of purification revealed that there was only a trace amount of N-OH-2-AAF sulfotransferase present in the female rat liver.

Animals↗

PubMed1976

A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic and spectrophotometric properties.

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IFO 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000). Stoichiometrical studies and the results obtained with 18O2 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively dentrified. The enzyme, which exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe3+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe2+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity. The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, 8-hydroxyquinoline, reducing agents such as 2-mercaptoethanol, and HgCl2. The Michaelis constants are as follows: 2-nitropropane (2.13 X 10(-2) M), nitroethane (2.43 X 10(-2) M), 3-nitro-2-pentanol (6.8 X 10(-3) M), 1-nitropropane (2.56 X 10(-2) M), and oxygen (3.03 X 10(-4) M, with 2-nitropropane).

Amino Acids↗

PubMed1977

The purification and characterization of rat liver lysosomal alpha-L-fucosidase.

The alpha-L-fucosidase from rat liver lysosomes was purified approximately 27,000-fold (from cytoplasmic extract) by a rapid procedure requiring only 7 h anf providing enzyme in a 20 per cent yield. The procedure is based upon affinity chromatography with agarose-epsilon-aminocaproyl-fucosamine. The isolated enzyme was found to be pure by a number of different analytical gel techniques and is essentially free of other lysosomal gylcosidases. The purified enzyme exhibits a positive periodic acid-Schiff stain, suggesting that it is a glycoprotein. The purified enzyme has a pH optimum of 5.7 to 5.9, a Vmax of 27 mumol/min/mg of protein, and a Km of 0.19 mM with p-nitrophenyl alpha-L-fucopyranoside as substrate. L-Fucose was the only possibly physiological effector of the enzyme which was identified; it exhibited a Ki of 1.6 mM, with p-nitrophenyl alpha-L-fucopyranoside as substrate. The enzyme has a subunit molecular weight of approximately 55,000 by Na dodecyl-SO4 electrophoresis in a variety of gel systems. The molecular weight of the native enzyme was indicated to be approximately 160,000 by sucrose density centrifugation, 300,000 by molecular sieve chromatography on Sephadex G-200, and 217,000 by sedimentation equilibrium centrifugation. The weight of evidence suggests that the enzyme is a tetramer. Incubation on the absence of sulfhydryl reagents under appropriate conditions generates a second alpha-L-fucosidase activity band on gels corresponding to a molecular weight of approximately 40,000 to 50,000. This result suggests that the subunit is relatively stable and may reassociate to form active enzyme. Alpha-L-Fucosidase requires a high concentration of protein and the presence of a sulfhydryl reagent for stabilization. It is rapidly inactivated by p-chloromercuriphenyl sulfonic acid, this inactivation being rapidly reversible by the addition of 10 mM 2-mercaptoethanol. The enzyme catalyzed the hydrolysis of 1 leads to 2, 1 leads to 3, and 1 leads to 4 fucosyl linkages and was found to be active on glycopeptides but not on native glycoproteins. The amino acid and carbohydrate composition of the enzyme was determined. The native enzyme contains the following sugars (residues per tetramer): fucose (3.5), mannose (32), galactose (8), glucose (9), glucosamine (32), and sialic acid (8). Rat liver lysosomal alpha-glucosidase, also produced in the rapid isolation procedure described herein, contained less than 0.1 residue of sialic acid per subunit.

Amino Acids↗

PubMed1976

Identification of albumin as the serum factor essential for the growth of activated human lymphocytes.

Albumin from human, bovine, or rabbit serum supported the growth of concanavalin A-stimulated human thymus-derived lymphocytes equally well. This activity was completely abolished by pepsin digestion. It was shown for bovine serum albumin that the albumin molecule itself, and neither an impurity nor a factor bound to albumin was essential for the growth of lymphocytes. This conclusion was based on observations that the growth-promoting activity could not be removed from albumin, and that the specific activity of albumin remained unaltered after the following procedures: molecular sieving at pH 7.5 at pH 3.0, and in 8 M urea at pH 6.6; ion exchange chromatography at pH 4.3 and in 8 M urea at pH 7.2; isoelectric focusing; charcoal treatment; acetone precipitation; and reduction with 2-mercaptoethanol in the presence of 8 M urea. Dimeric albumin was found to support growth of lymphocytes as well as monomeric albumin, and mercaptalbumin and non-mercaptalbumin were shown to have equal activity.

Animals↗

PubMed1975

Decarboxylation of oxalacetate to pyruvate by purified avian liver phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase, which has been isolated from chicken liver mitochondria in essentially homogenous form, carries out the irreversible decarboxylation of oxalacetate to pyruvate in the presence of catalytic amounts of GDP or IDP, as well as the reversible decarboxylation of oxalacetate to phosphoenolpyruvate in the presence of substrate amounts of GTP or ITP. The pyruvate- and phosphoenolpyruvate-forming reactions are similar in their nucleoside specificity and appear to be carried out by the same protein. However, the two activities vary markedly in their response to added metal ions and sulfhydryl reagents. Phosphoenolpyruvate formation is completely dependent on the presence of a divalent metal ion, with Mn2+ the most effective species. This reaction is also stimulated by sulfhydryl reagents such as 2-mercaptoethanol. In contrast, the pyruvate-forming reaction is strongly inhibited by divalent metal ions, including Mn2+, and also by moderate concentrations of sulfhydryl reagents. These observations and the demonstration that pyruvate kinase-like activity is very low or absent make it unlikely that pyruvate formation proceeds via phosphoenolpyruvate as an intermediate. Although the pyruvate-forming reaction is inhibited by added metal ions, the reaction is also inhibited by metal-chelating agents such as 8-hydroxyquinoline and o-phenanthroline, suggesting that the reaction is dependent on the presence of a metal ion. It has not been possible, however, to demonstrate that the enzyme is a metalloprotein.

2,2'-Dipyridyl↗

PubMed1975

Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus.

Inorganic pyrophosphatase [EC 3.6.1.1] was purified from Bacillus stearothermophilus to a homogeneous state both ultracentrifugally and electrophoretically. Ultracentrifugal analysis revealed that the molecular weight of the enzyme is 122,000 and the sedimentation coefficient (S0.34%/20, W) is 5.2S. The enzyme molecule in 0.1% sodium dodecylsulfate solution containing 1 mM 2-mercaptoethanol had an estimated molecular weight of 70,000 on the basis of SDS-polyacrylamide gel electrophoresis results, which indicates that the enzyme may consist of two subunits. Divalent cations such as Mg2+, Mn2+, and Co2+ are required for the enzymatic activity. Pyrophosphate is the only substrate for the enzyme. ATP and p-chloromercuribenzoate inhibit the enzyme reaction markedly.

Adenosine Triphosphate↗