Kinin-releasing enzyme from the venom of Bitis arietans (puff adder)

A kinin-releasing enzyme was isolated from Bitis arietans (puff adder) venom by Sephadex G-100 and DEAE-cellulose column chromatographies. The kinin-releasing enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion. Its molecular mass is approximately 45 kDa with an isoelectric point of 6.5. Kinin-releasing enzyme possesses proteolytic activity which hydrolyzes the Leu⁶-Cys⁷, His¹⁰-Leu¹¹ and Ala¹⁴-Leu¹⁵ bonds of the B chain of oxidized insulin and the Aα and Bβ chain of fibrinogen. Kinin-releasing and benzoyl-l-arginine ethyl ester hydrolytic activities of this enzyme were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is involved in enzymatic activities.     

Purification and properties of sucrose synthetase from morning-glory callus cells

Sucrose synthetase was purified about 130-fold from morning-glory (Pharbitis nil Choisy cv. Murasaki) callus cells, and the properties of sucrose synthesis and cleavage activities of the enzyme were compared. The enzyme preparation gave a single band by disc electrophoresis. The molecular mass of the enzyme was estimated to be 4.2 × 10⁵ by gel filtration. The enzyme preparation gave two bands by SDS disc electrophoresis, suggesting the molecular mass of about 3.8 ×10⁴ and 7.0 × 10⁴. The pH optima of sucrose synthesis and cleavage activities of the enzyme were different from each other, giving pH 9.0 and pH 6.5 respectively. MgCl², MnCl² and CaCl² activated the sucrose synthesis activity about two times the normal rate and conversely inhibited the sucrose cleavage activity. F-6-P was not replaced by fructose. UDP was the only valuable substrate as a nucleotide diphosphate. The enzyme showed the negative ecoperativity effect of UDPG suggesting to be an allosteric enzyme. The Km values of sucrose and fructose were calculated to be 167 mM and 5 mM, respectively. UDP suggested substrate inhibition. The apparent equilibrium constant varied between 1 to 3. Based on these results, the role of the enzyme in the sucrose metabolism of morning-glory callus cells will be discussed.     

Apurinic acid endonuclease implicated in DNA breakage in Escherichia coli subjected to mild heat.

Nicotinamide nucleotide transhydrogenase from beef heart mitochondria was purified to homogeneity and characterized. The enzyme is devoid of other respiratory chain activities as well as flavin. Reduction of NAD⁺ by NADPH catalyzed by reconstituted transhydrogenase generates an uncoupler-sensitive uptake of lipophilic anions, whereas the rate of reduction of NAD⁺ by NADPH is enhanced about 13 fold by uncouplers. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate reveales that the protein consists of a single polypeptide of a molecular weight of 97,000.     

Purification and properties of an ld-dipeptidase FROM Bacillus sphaericus

• 1.1. An ld-dipeptidase (EC 3.4.13.-) that hydrolyzes the unrelated dipeptides l-Ala-d-Glu (sp. act. 0.85 μmol·min⁻¹·mg⁻¹) and l-Lys-d-Ala (sp. act. 11 μmol · min⁻¹·mg⁻¹) has been purified 250-fold from the sporulation medium of Bacillus sphaericus with a 4% recovery of lytic activity.
• 2.2. Throughout the purification steps, followed with both substrates, the enzyme peaks of activities were congruent and the ratios of activities were constant. Both activities were activated 50-fold by cobalt. Polyacrylamide gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. The data are consistent with those activities being due to a single enzyme.
• 3.3. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band (M_r 38,000).
• 4.4. This dipeptidase hydrolyzes some other ld-dipeptides with a free amino and carboxyl group. Although dipeptides having a di-amino acid as the amino terminus are the best of the substrates tested, the hydrolysis occurs also when neutral amino acids are N-terminal. The activity is higher with neutral C-terminal residues such as Gly or d-Ala than with a di-acid residue such as d-Glu.
• 5.5. This enzyme may have a function in peptidoglycan metabolism.

     

Transport ATPase: further studies on the properties of the thimerosal-treated enzyme.

Our previous studies showed that when ethylmercurithiosalicylate (thimerosal) interacts with the transport ATPase of the guinea pig kidney under specified conditions, the Na⁺ + K⁺-dependent ATPase activity is inhibited, while the Na⁺-dependent ATPase, the Na⁺ + ATP-dependent phosphorylation of the enzyme, and the K⁺-dependent discharge of the phosphoenzyme seem to be unaffected. Here we describe other properties of the thimerosal-treated enzyme: Na⁺-dependent ADP-ATP exchange, Na⁺-dependent UTPase, and K⁺-dependent p-nitrophenylphosphatase activities of the modified enzyme are not inhibited. Kinetics of the Na⁺ effect on the UTPase activities of the native and the modified enzyme are the same. However, K⁺ has a greater inhibitory effect on the Na⁺-UTPase of the modified enzyme than on the Na⁺-UTPase of the native enzyme. The increase in the apparent affinity of the thimerosal-treated enzyme for K⁺ is also evident from the kinetics of the K⁺ effect on p-nitrophenylphosphatase. Neither the native enzyme nor the modified enzyme catalyzes a P₁-ATP exchange. The uninhibited activities of the thimerosal-treated enzyme are sensitive to ouabain. These data provide further support for those reaction mechanisms in which the existence of two ATP sites within the enzyme is assumed.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.     

Metabolic regulation of malic enzyme activity from Paracoccus denitrificans by glyoxylate and acetylCoA.

$\text{Paracoccus denitrificans}$ contains both NAD⁺- and NADP⁺-linked malic enzyme activities when grown on malate/nitrate. The enzyme is inactive in the absence of NH₄⁺. AcetylCoA inhibits both activities competitively with respect to L-malate. Glyoxylate (0.5 mM) causes 60% inhibition of the NADP⁺-linked activity but has little effect on the NAD⁺-linked activity. Citrate, aspartate, AMP, ADP, and ATP, at 0.5mM, have little effect on either of the two activities. The results are discussed with regards to the control of malic enzyme activity within the cell.     

The localization of galactosyltransferases in polyacrylamide gels by a coupled enzyme assay

A coupled enzyme assay for GlcNAc¹: UDP-galactose galactosyltransferase has been developed that allows this enzyme to be assayed spectrophotometrically and in nondenaturing polyacrylamide gels. Utilizing three, intermediate enzymes, galactosyltransferase activity has been coupled to the production of NADH with a stoichiometry of 2 mol of NADH produced for each mol of galactose transferred to GlcNAc. The enzyme reactions coupled to the production of UDP by galactosyltransferase can be summarized as follows:

The activities of partly purified bovine milk galactosyltransferase and galactosyltransferase in dialyzed fetal calf serum have been determined spectrophotometrically by measuring NADH production at 340 nm. The reaction is dependent on N-acetylglucosamine, UDP-galactose, and Mn²⁺. For both enzyme sources, activities calculated from NADH production are similar to those determined from assays that use radioactive sugar nucleotide substrates. Both galactosyltransferase activities have been localized on 7.5% nondenaturing polyacrylamide gels after electrophoresis by incubating the gel with an agarose indicator gel containing the coupled enzyme system. Enzyme activity is marked by NADH fluorescence, which is dependent on the presence of N-acetylglucosamine in the indicator gel. The intensity of fluorescence increases with increasing galactosyltransferase activity applied to the gel.     

Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD⁺-dependent dehydrogenase (type D) and as the O₂-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD⁺ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K_m for xanthine is not significantly different for the two forms.     

Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase

A single enzyme catalyzes the synthesis of all four N²-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N²-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (K_m) differ, the ratio V/K_m is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Effects of molybdenum and tungsten on induction of nitrate reductase and formate dehydrogenase in wild type and mutant Paracoccus denitrificans

Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein M _r150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein M _r150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wilde type. Autoradiography of preparations from cells incubated with ⁵⁵Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with ⁹⁹Mo, incorporation of molybdenum into the mutant protein was not detectable.We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.Non-Standard Abbreviations DOC deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the ¹H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T₂, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the ¹H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by ¹H n.m.r. spectroscopy.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Metal stoichiometry, coenzyme binding, and zinc and cobalt enchange in highly purified yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase, purified from baker's yeast under conditions which exclude contamination by extraneous metal ions, is homogeneous by analytical ultracentrifugation and disc gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a molecular weight of 149,000 as determined by ultracentrifugation time-lapse photography and exhibits specific activities of 430 to 480 U/mg. Zinc analysis by three independent, highly sensitive methods, i.e., atomic absorption spectrometry, atomic fluorescence spectrometry, and microwave-induced plasma emission spectrometry, demonstrates 4 g-atom of catalytically essential Zn per mole of enzyme. No other metal atoms are present in stoichiometrically significant quantities as assessed by emission spectrography. The Stoichiometry of coenzyme binding, 4 mol of NADH/mol of enzyme, is identical to that of zinc, consistent with one coenzyme binding site and one zinc atom per enzyme subunit. Conditions for exchange of the four catalytically essential zinc atoms with ⁶⁵Zn have been developed. These atoms exchange identically under all conditions examined. The resultant radiolabeled enzyme, l(YADH)⁶⁵Zn₄], has the same metal content, specific enzymatic activity, and coenzyme binding properties as the native enzyme. The ⁶⁵Zn of this enzyme serves to monitor the extent and site specificity of cobalt replacement. The fully cobalt-substituted enzyme, [(YADH)Co₄], has a specific activity of 80 U/mg, 17% that of the Zn enzyme, and exhibits absorption and circular dichroic spectra which are consistent with coordination by one or more sulfur ligands in a distorted tetrahedral geometry.     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane

Mouse leukemia L-1210 cells were iodinated with ¹²⁵I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of ¹²⁵I over that of the cell homogenate and a 20-fold increase in the specific activities of 5′-nucleotidase and alkaline phosphate; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide ($\text{M}{}_{\mathrm{<mtext>m</mtext>}}$ 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel sytem for the analysis of membrane proteins is emphasized.     

Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes

The proteins of Sarcoplasmic reticulum membranes were resolved by polyacrylamide gel electrophoresis into several fractions ranging in mol wt from 300,000 to about 30,000. The ATPase enzyme involved in Ca²⁺ transport is associated with a major protein fraction and its molecular weight based on its electrophoretic mobility on polyacrylamide gels in the presence of sodium dodecylsulfate is about 106,000. Reducing agents (β-mercaptoethanol or dithiothreitol) cause the dissociation of membrane proteins into subunits of 20,000–60,000 mol wt, which can be separated by electrophoresis or Sephadex G-150 chromatography.     

Phosphohistidine as a stoichiometric intermediate in reactions catalyzed by isoenzymes of wheat germ acid phosphatase.

Burst titration experiments conducted on a highly purified isoenzyme of wheat germ acid phosphatase under conditions where [S]_o > K_m indicate that there is one titratable active site per molecule of enzyme of molecular weight 59,000. The enzyme is labeled to only a small extent with inorganic [³²P]phosphate ion. Incubation of wheat germ acid phosphatase with ³²P-labeled substrates such as p-nitrophenyl phosphate or inorganic pyrophosphate followed by quenching in alkali results in the stoichiometric trapping of a base-stable, acid-labile phosphorylated protein. The extent of ³²P incorporation parallels the degree of purity of the enzyme and corresponds to the incorporation of 1 mol of phosphate per mole of enzyme. The incorporation is eliminated by the simultaneous presence of excess unlabeled phosphate ion (a competitive inhibitor) and is not observed when a noncatalytic protein (such as bovine serum albumin) is substituted for the enzyme. Complete alkaline hydrolysis of the labeled protein results in the recovery of an 85% yield of τ-phosphohistidine, identified by ion-exchange chromatography, high-voltage paper electrophoresis, and comparison with a synthetic sample. A ³²P-labeled tryptic tetradecapeptide was isolated following hydrolysis of the labeled, reduced, and carboxymethylated protein with trypsin at pH 8.3, separation of the labeled peptide, and purification by two methods including a novel variant of a diagonal electrophoresis technique. The end groups and composition of the peptide are reported. The data are consistent with the interpretation that a phosphohistidine-enzyme intermediate is formed as an obligatory intermediate in the catalytic reaction involving this enzyme.     

Purification and properties of a homogeneous aryl sulfatase A from rabbit liver.

Aryl sulfatase A (aryl sulfate sulfohydrolase EC 3.1.6.1) has been purified > 10,000-fold from rabbit liver; by disc gel electrophoresis the enzyme appears homogeneous. Various properties of the enzyme have been determined and comparisons are made with other aryl sulfatases. Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is made up of monomers of molecular weight ～ 70,000. At pH 7.4 the enzyme exists as a dimer whereas a tetrameric form predominates at pH 4.8.The enzyme exhibits the anomalous kinetics often observed with aryl sulfatase A from mammalian tissues (the enzyme is modified to an inactive form while degrading substrate and the inactive form can be reactivated by sulfate ion). The enzyme activity has been studied under a variety of reaction conditions. Two pH optima are observed and neither enzyme concentration or changes in ionic strength appear to have an effect on the relative magnitudes of the optima. Aryl sulfatase A is competitively inhibited by potassium sulfate, potassium phosphate, and sodium sulfite (K_i = 2.9 × 10^?3 M, 3.4 × 10^?5 M, and 1.1 × 10^?6 M, respectively). Kinetic constants for some substituted phenyl sulfate esters have been determined. The variation in V is not consistent with a reaction mechanism involving a rate-limiting breakdown of a common intermediate.The inactive (modified) form of the enzyme has been isolated from reaction mixtures containing aryl sulfatase A and substrate. A procedure is presented for determining the relative amount of modified and native enzyme in these preparations. In the presence of substrate, sulfate displaces the equilibrium between native and modified enzyme in favor of native enzyme. In the absence of substrate neither sulfate or phosphate have an effect on the equilibrium. A study is made of the temperature dependence of the process in which the modified enzyme is converted back to native enzyme. The relatively small entropy of activation for the conversion of the modified to the native form (ΔS3 = ?8 cal/mole deg) does not seem to be consistent with a major modification of protein conformation.     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.