Properties of Crystalline α-Glucosidase from Mucor javanicus

Substrate and inhibitor specificities, and transglucosylation action of crystalline α-glucosidase from the mycelia of Mucor javanicus have been investigated. The enzyme hydrolyzed maltose, methyl-α-maltoside, and soluble starch liberating glucose, but little or not phenyl-α-glucoside, methyl-α-glucoside, sucrose, isomaltose, panose and dextran. The enzyme hydrolyzed phenyl-α-maltoside to glucose and phenyl-α-glucoside. The enzyme acted also as a glucosyltransferase when it was incubated with glucosyl donor such as maltose. Maltotriose was the principal transglucosylation product formed from maltose. The enzyme also catalyzed transglucosylation from maltose to riboflavin, pyridoxine, esculin and rutin. Tris and turanose inhibited the enzyme activity, but PCMB and EDTA did not. It is suggested that the enzyme activity is closely related to the histidine residue in the active center, from the inhibition experiments using diazonium-1-H-tetrazole and rose bengal.     

Thiol groups of chicken liver LDH (author's transl)]

Crystallized chicken liver H4 lactatedehydrogenase with PCBM and DTNB, proved to have sic thiol groups per enzyme molecule. Sulphydryl groups seemed necessary for activity since the enzyme became inactive when the groups were blocked by PCMB, DTNB or by Zn (II), Cu (II) or Hg (II). LDH inhibited by Hg (II) recovered its activity after treatment with beta-mercaptoentanol. LDH reversible inactivation, caused by PCMB, was partially impeded by NAD, NADH hand L-lactate but inactivation caused by DTNB was impeded in any way by coenzymes or substrates. PCMB is a competitive inhibitor with the coenzymes but is non-competitive with the substrates whereas DTNB is a competitive inhibitor with NADH or L-lactate. Kinetic studies of the DTNB inactivation suggest the possible formation of a DTNB-LDH-NADH complex. The formation of LDH-NADH and LDH-NAD pyruvate inactive complexes have been detected by U.V. absorbancy measurements. Such inactive complexes have equally been observed experimenting with the PCMB of Hg (II) previously treated enzyme. The results showed that these essential sulphydryl groups are not involved in th attaching of coenzymes or substrates to the chicken liver LDH molecule, but they seem to suggest the participation of --SH groups during the reversible hydrogen transfer between NADH and pyruvate.     

Some Characteristics of Formyltetrahydrofolate Synthetase from Pea Seedlings

It has been found, that ammonium sulfate is effective not only in stabilizing, but also in stimulating the activity of formyltetrahydrofolate synthetase (E. C. 6, 3. 4. 3) purified approximately 500-fold from pea seedlings. Kinetic studies have indicated that the stimulation by ammonium sulfate is due to the enhancement of the binding of the substrate, formate, with the enzyme. The binding of the another substrate, FAH₄, with the enzyme was not affected by the addition of ammonium sulfate. The enzyme activity was inhibited by various sulfhydryl reagents, and the inhibition by PCMB was overcome by the addition of l-cysteine. The inhibition by PCMB was competitive with FAH₄, and the K_i value for PCMB was 0.8 × 10?⁶m.     

The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme

1. The ;xanthine oxidase' activity of rat liver supernatant, most of which behaves as an NAD(+)-dependent dehydrogenase (type D) can be rapidly converted into an oxidase (type O) by thiol reagents such as tetraethylthiuram disulphide, copper sulphate, 5,5'-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercuribenzoate. Treatment with copper sulphate, if prolonged, leads to almost complete inactivation of the enzyme. The effect of these reagents is prevented by dithioerythritol, and in all cases but that of N-ethylmaleimide is reversed by the same thiol. 2. Dithioerythritol prevents and reverses the conversion of xanthine oxidase from type D into type O brought about by storage of rat liver supernatant at -20 degrees C, preincubation under anaerobic conditions, treatment with carbon or with diethyl ether, and reverses, but does not prevent, the conversion obtained by preincubation of the whole liver homogenate. 3. Conversion of the enzyme from type D into type O is effected by preincubation of rat liver supernatant with the sedimentable fraction from rat liver but not from chick or pigeon liver. The xanthine dehydrogenase activity of chick liver supernatant is not changed into an oxidase by preincubation with the sedimentable fraction from rat liver. 4. The enzyme activity of rat liver supernatant is converted from type D into type O during purification of the enzyme: the purified enzyme can be reconverted into type D by dithioerythritol. 5. The enzyme appears as an oxidase in the supernatant of rat heart, intestine, spleen, pancreas, lung and kidney. The enzyme of all organs but intestine can be converted into a dehydrogenase by dithioerythritol.     

Effects of the photobleaching herbicide, acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles, soybean root mitochondria, soybean root nodules, and bacteria

The photobleaching herbicide, acifluorfen-methyl (AFM), has been reported to be an inhibitor of the heme and chlorophyll biosynthetic enzyme protoporphyrinogen oxidase (Protox) in several plant species. However, AFM had no effect on the levels of Protox activity measured in a mitochondrial fraction from soybean roots. In contrast, AFM inhibited Protox activity in etioplasts from barley leaves and in mitochondria from barley roots, but the extent of inhibition varied depending upon the assay conditions and was maximal only in the presence of 5 mM dithiothreitol (DTT). AFM inhibition was enhanced by preincubation of barley organelle extract in the presence of DTT. Preincubation of barley extract with DTT and AFM together (but not with AFM alone) caused extensive enzyme inhibition which was not reversible by dialysis. These findings have implications for the mechanism of AFM action and for the differential effect of these herbicides on crop and weed species. AFM had no effect on the Protox activity of membranes from free-living bacterial cell of Bradyrhizobium japonicum or Escherichia coli, or on the high levels of Protox activity associated with the plant-derived membrane surrounding the symbiotic bacteria within the soybean root nodule.     

Mechanism of inhibition of human neutrophil collagenase by Gold(I) chrysotherapeutic compounds. Interaction at a heavy metal binding site

The mechanism of inhibition of two forms of human neutrophil collagenase (HNC) by six Au(I) compounds, some of which are used as chrysotherapeutic agents, has been investigated. The two forms of enzyme studied are active and latent HNC, the latter of which is activated by p-chloromercuribenzoate (PCMB). The effects of PCMB and Zn(II), which are normally included in the assays, on the activity of both forms of HNC and on their inhibition by these Au(I) compounds have also been studied. Zn(II) stimulates the activity of both the active and PCMB-activated latent forms of HNC up to a concentration of 50-100 microM, after which it inhibits markedly. PCMB activates latent HNC up to a concentration of 100 microM followed by inhibition at higher concentrations. Active HNC is not stimulated at PCMB concentrations below 100 microM, but is inhibited at higher concentrations. The stimulatory effects of Zn(II) and PCMB on HNC and its inhibition by PCMB are all attributable to binding at distinct sites. The inhibition of both active and PCMB-activated latent HNC by the Au(I) compounds is noncompetitive and is reversed by Zn(II). The inhibition of both forms of HNC by SKF 80544 and SKF 36914, which do not contain thiol ligands, is weak to moderate and is not influenced by the PCMB concentration. In contrast, PCMB markedly enhances the inhibition by Myocrisin, Sanocrisin, and Solganol by complexing to their thiol ligands to facilitate release of the Au(I) atom for binding to HNC. Cd(II) and Cu(II) also inhibit HNC noncompetitively, and inhibition is also reversed by Zn(II). Collectively, these data indicate that latent HNC contains a heavy metal binding site distinct from the active site at which Au(I), Cd(II), and Cu(II) bind to cause noncompetitive inhibition. Occupancy of this site by Zn(II) is characterized by retention of activity.     

Reversible dissociation of cortisol-transcortin complex by sodium para-chloromercuribenzoate

Mercurials are considered as sulphydryl group specific reagents and one of them, sodium para-chloromercuribenzoate (PCMB), is currently used for SH titration. It has been shown that cellular steroid receptors are reversibly inactivated by mercurials even when the binding site is occupied by the steroid (Coty, W.A. (1980) J. Biol. Chem. 255, 8035-8037). This is a striking difference with alkylating SH reagents such as iodoacetic acid or N-ethylmaleimide, since these reagents inactivate only steroid-free receptors. In order to explain this discrepancy, we tested, in the present study, the specificity of PCMB on a blood plasma steroid binding protein: human transcortin. This protein presents the advantage, over cellular receptors, of being well characterized and to be available in a pure state. The transcortin-cortisol complex was also reversibly inactivated by PCMB when the reaction was carried out at a high excess of reagent over protein; such conditions are those previously used with steroid receptors. The reversibility was obtained not only with a reducing agent (dithiothreitol) but also with EDTA, which suggests a poor stability of the protein mercurial bond and therefore a nonspecific action. The decrease of activity was the result of a loss of binding sites and Scatchard plot analysis did not reveal any detectable decrease of the affinity constant for cortisol. Transcortin possesses two SH groups per molecule, one of these being buried in native conformation. After blockage of the accessible SH group by aminoethylation, transcortin kept the same activity, but when this aminoethylated transcortin was incubated with PCMB a loss of activity was obtained, although the residual buried SH group was again titrable with Ellman's reagent. Therefore, we can conclude that the action of PCMB on proteins must be interpreted with precaution, since it can induce an inactivation that is SH-independent.     

A persistent active state of the adenylate cyclase system produced by the combined actions of isoproterenol and guanylyl imidodiphosphate in frog erythrocyte membranes.

Isoproterenol plus guanylyl imidodiphosphate (Gpp(NH)p) activate frog erythrocyte adenylate cyclase to a level much higher than the sum of the activities produced by the catecholamine and the synthetic nucleotide tested separately. Propranolol, the beta-receptor blocking agent, failed to inhibit activity when added after the enzyme system had been preincubated with both isoproterenol and Gpp(NH)p. However, if propranolol was added after only one of the two components had been added, it inhibited the effect of isoproterenol. Production of the propranolol-resistant state by treatment with isoproterenol and Gpp(NH)p did not require the presence of the productive substrate (MgATP). The activated propranolol-resistant state persisted following various treatments of the enzyme preparation including extensive washings of the membranes; considerable activity was retained even after sonication or treatment with the detergent Lubrol-PX, treatments which caused inactivation of the native enzyme. Extensive dilution of the membranes following pretreatment with isoproterenol and Gpp(NH)p did not significantly reduce to the activity of the enzyme. Readdition of isoproterenol after dilution caused some inhibition of adenylate cyclase activity, indicating apparently that the beta-receptor has not become inaccessible. In contrast, preincubation with isoproterenol alone failed to render the enzyme system refractive to propranolol, and dilution readily reduced the activity to negligibly low values. Preincubation with Gpp(NH)p alone also produced a persistent active state but the activity was much lower than that obtained throught the combined action of isoproterenol and Gpp(NH)p. The findings suggest that the hormone may be required only to facilitate the initial interaction of the enzyme with Gpp(NH)p. The differences, in this respect, between Gpp(NH)p and the more labile natural nucleotide, GTP, are discussed.     

Effect of thiol reagents on the activity of NADP-dependent malate dehydrogenase isolated from bovine adrenal cortex cytoplasm

NADP-dependent malate dehydrogenase was rapidly inactivated in the presence of mercurous chloride. Titration of malate dehydrogenase by 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in a solution of 8 M urea revealed 18 SH groups per molecule of the enzyme. Eight sulphydryl groups reacted with DTNB in native malate dehydrogenase and their modification was not accompanied by a loss of the enzyme activity. The interaction of p-chloromercury benzoate (PCMB) with malate dehydrogenase resulted in a 70% decrease in the enzyme activity. The binding of the thiol reagents by the malate dehydrogenase molecule appreciably increased the Michaelis constant value for the substrate. In the presence of magnesium ions, NADP and malate did not affect the process of malate dehydrogenase modification by DTNB and did not protect the enzyme from the inactivation by PCMB. It is suggested from the data obtained that the sulphyryl groups are involved in maintaining the active conformation of the enzyme.     

Isolation, purification, and characterization of a new enzyme from Pseudomonas sp. M-27, carboxypeptidase G3.

A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60,000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (M(r): 15,000). The enzyme hydrolyzed predominantly acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles carboxypeptidase G1 or G2 in its glutamate-releasing activity. However, it acts not only on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G1 or G2, it was named carboxypeptidase G3.     

Purification and characterization of phosphodiesterase from the venom of Trimeresurus mucrosquamatus

Phosphodiesterase was isolated from the venom of Trimeresurus mucrosquamatus from Taiwan using gel filtration on a Sephadex G-100 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and immunodiffusion. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB) but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approximately 140,000 and the isoelectric point was found to be pH 7.4 by isoelectric focusing with carrier ampholyte. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 5.6 X 10(-3) and 7.6 X 10(-4) M, respectively.     

Purification and regulation of aspartate transcarbamylase from germinated mung bean (Vigna radiata) seedlings

Aspartate transcarbamylase (EC 2.1.3.2) was purified to homogeniety from germinated mung bean seedlings by treatment with carbamyl phosphate. The purified enzyme was a hexamer with a subunit molecular weight of 20,600. The enzyme exhibited multiple activity bands on Polyacrylamide gel electrophoresis, which could be altered by treatment with carbamyl phosphate or UMP indicating that the enzyme was probably undergoing reversible association or dissociation in the presence of these effectors. The carbamyl phosphate stabilized enzyme did not exhibit positive homotropic interactions with carbamyl phosphate and hysteresis. The enzyme which had not been exposed to carbamyl phosphate showed a decrease in specific activity with a change in the concentration of both carbamyl phosphate and protein. The carbamyl phosphate saturation and UMP inhibition patterns were complex with a maximum and a plateau region. The partially purified enzyme also exhibited hysteresis and the hysteretic response, a function of protein concentration, was abolished by preincubation with carbamyl phosphate and enhanced by preincubation with UMP. All these observations are compatible with a postulation that the enzyme activity may be regulated by slow reversible association-dissociation dependent on the interaction with allosteric ligands     

The generation of big-endothelin (1-22) (endothelin-valine) from big-endothelin in the soluble fraction of porcine lung.

The degradation of big-endothelin (big-ET) in the soluble fraction of porcine lung was investigated. The degradation in the presence of p-chloromercuribenzoate (PCMB), pepstatin A, and EDTA resulted in the accumulation of two newly-formed fragments, big-ET (23-39) and big-ET (1-22), the latter called endothelin-valine (ET-Val). The generation of the two fragments was inhibited by diisopropylfluorophosphate (DFP). The enzyme responsible, called ET-Val-generating endopeptidase, was isolated from porcine lung by a procedure including chromatographies on columns of DEAE-cellulose, hydroxylapatite, Mono Q, p-mercuribenzoate-Sepharose, and Superose 6. The molecular weight of the enzyme was 140,000 and the pH optimum of the activity was 7.0. The activity was strongly inhibited by DFP, but scarcely inhibited by PCMB, EDTA, and pepstatin A. Thus, the isolated enzyme was classified as a serine protease cleaving big-ET at the Val22-Asn23 bond.     

Phosphodiesterase from the venom of Crotalus ruber ruber

Phosphodiesterase was isolated from the venom of Crotalus ruber ruber from the U.S.A. using the gel filtration on a Sephadex G-75 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and isoelectric focusing electrophoresis. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB), but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approx. 98,000 and the isoelectric point was found to be pH 10.5 by isoelectric focusing with carrier ampholyte. This enzyme contained 1.04 mol zinc per mol. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 8.3 X 10(-3) and 1.2 X 10(-2) M, respectively.     

粘质赛氏菌胞外蛋白酶的化学修饰

用九种化学修饰剂研究了粘质赛氏菌ＳｅｒｒａｔｉａＭａｒｃｅｓｃｅｎｓ４１００３（２）胞外蛋白酶分子中氨基酸侧链基团与酶催化活性的关系,结果表明组氨酸、丝氨酸、赖氨酸、精氨酸、谷氨酸及天冬氨酸等残基与酶活性无关;半胱氨酸残基与酶活性也无直接关系;而酪氨酸和色氨酸残基侧链的修饰引起酶活力大幅度下降,说明酪氨酸和色氨酸残基为酶活力必需．     

Histochemical studies on Raillietina (Raillietina) johri (Cestoda: Davaineidae). II. Nucleoside diphosphatase and thiamine pyrophosphatase.

Thiamine pyrophosphatase and nucleoside diphosphatase have been studied histochemically in Raillietina (Raillietina) johri. Thiamine pyrophosphatase activity has been observed in the tegument, subtegumental muscle, subtegumental cells, medullary parenchyma, excretory canal and various reproductive structures like testes, ovary, vas deferens, spermatozoa and vitellaria. Eggs exhibit moderate enzyme activity. Various nucleoside diphosphates have been found to be hydrolyzed by thiamine pyrophosphatase. CaCl2, MgCl2 and MnCl2 each activated the enzyme at a final concentration of 6 mM whereas cysteine, reduced glutathione and PCMB inhibited the enzyme activity at a final concentration of 10 mM, 10 mM and 20 mM, respectively. KCN and NaF had no effect on the enzyme staining at concentration as high as 50 mM and 30 mM, respectively. Possible roles of the enzyme in the parasite have been discussed.     

Certain Properties of α-Glucosidase from Mucor racemosus

The substrate and inhibitor specificities, and α-glucosyltransfer products of the purified α-glucosidase from the mycelia of Mucor racemosus were investigated. The enzyme hydrolyzed maltose, maltotriose, phenyl α-maltoside, isomaltose, soluble starch, and amylose liberating glucose, but did not act on sucrose. The enzyme hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. Maltotriose was the main a-glucosyltransfer product formed from maltose, and isomaltose was that from soluble starch. Tris and turanose inhibited the enzyme activity, but PCMB and EDTA did not. The enzyme hydrolyzed amylose liberating a-glucose. The enzyme was a glycoprotein containing 4.1% of neutral sugar. The neutral sugar was identified as mannose in the acid hydrolyzate of the enzyme.     

Cysteine desulfhydrase activity in cucurbit plants: Stimulation by preincubation with l- or d-cysteine

Cysteine desulfhydrase activity in leaf discs of cucurbit plants is enhanced 2–4-fold by preincubation with l or d-cysteine. Preincubation with structural analogs of cysteine also stimulated the activity of the enzyme, but to a smaller extent. Maximal increase in cysteine desulfhydrase activity was observed by preincubation with 5 mM or higher concentrations of cysteine. Although not caused by activation, stimulation of the enzyme activity was half-maximal within less than 15 min. Whereas the increase in cysteine desulfhydrase activity by preincubation of leaf discs with cysteine was light independent, pretreatment of the entire plant with light or dark determined the leaf discs' potential for stimulation of the enzyme. Exposure to darkness for 4 hr reduced this potential by 60%. It is concluded that the potential for stimulation of cysteine desulfhydrase activity by preincubation with cysteine is regulated by a compound not synthesized, but metabolized, in the leaf tissue. This regulatory compound may be supplied to the leaves by long-distance transport.     

Deacetylcephalosporin C Formation by Cephalosporin C Acetylhydrolase Induced in a Cephalosporium acermonium Mutant

Cephalosporin C acetyl-hydrolase, which had not yet been found in Cephalosporium acremonium cultures, was partially purified from the culture fluid of the mutant No. 81 by ammonium sulfate fractionation, dialysis and DEAE-cellulose column chromatography. The optimum pH and temperature of the enzyme reaction were found to be about 8.0 and 50°C, respectively. The enzyme activity was hardly affected by Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ni²⁺, Na⁺, K⁺, EDTA, PCMB and 2,4-dinitrophenol, but markedly inhibited by diisopropylfluoro-phosphate at 1 mm. The product formed from cephalosporin C by the enzyme reaction was proved to be deacetylcephalosporin C by physical and chemical analyses and chromatographic behaviors.