Lipase from Pseudomonas fragi. II. Properties of the Enzyme

The optimal pH value of a lipase from Pseudomonas fragi was between 7.5 and 8.9, and a high reaction rate was observed at 54 C. Heating the enzyme solution at 63 C for 30 min inactivated only 27.6% of its activity; however, total inactivation was observed at 66 C after 1 hr and at 71 C after 10 min. The lipase was inhibited strongly by Fe⁺⁺⁺ and Fe⁺⁺ ions, and to a lesser extent by Co⁺⁺, Cu⁺⁺, Zn⁺⁺. No inhibition was observed with Ca⁺⁺ or NaF. Ethylenediaminetetraacetate was effective in removing the toxicity of Fe⁺⁺⁺. The activity of the enzyme was inhibited markedly by p-chloromercurobenzoate, but the effects of N-ethylmaleimide and iodoacetate were moderate. The enzyme was able to hydrolyze natural fats, synthetic triglycerides, and alcohol esters. The order of the rate of hydrolysis of some triglycerides under experimental conditions was, from the fastest to the lowest, trilaurin, tricaprin, tricaprylin, tripalmitin, tributyrin, tricaproin, and tristearin. The enzyme was capable of hydrolyzing methyl butyrate, but the rate of hydrolysis was about one-fifth that for triolein and one-thirteenth that for coconut oil. The enzyme lost its activity rapidly when held frozen, at 20 C, and at the extremes in pH. Glutathione, cysteine, and mercaptoethanol did not preserve the activity of the enzyme.     

Ultracytochemical localization of Ca++-ATPase activity in pituicytes of the neurohypophysis of the guinea pig

Summary Ca⁺⁺-ATPase activity (cf. Ando et al. 1981) was examined both light- and electron-microscopically in the neurohypophysis of the guinea pig. Apart from a strong activity within the walls of the blood vessels, in the parenchyma of the neurohypophysis the reaction product of the Ca⁺⁺-ATPase activity was restricted to the plasmalemma of the pituicytes. This reaction was completely dependent upon Ca⁺⁺ and the substrate, ATP; the reaction was inhibited by 0.1 mM quercetin, an inhibitor of Ca⁺⁺-ATPase. A reduction of the enzyme activity occurred by 1) adding Mg⁺⁺ to the standard incubation medium, and 2) substituting Ca⁺⁺ with Mg⁺⁺ at varing concentrations. In all experiments the neurosecretory fibers were devoid of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity in the plasmalemma of the pituicytes is discussed in connection with the regulation of the extracellular Ca⁺⁺ concentration, which seems to be important with respect to the discharge of secretory material from the neurosecretory fibers.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany.     

Substrate Specificity and Some Properties of Crystalline Mold Maltase

The substrate specificity of crystalline mold maltase was investigated.

The enzyme acts upon various α-heteroglucosides or saccharides. Aryl-α-glucosides were hydrolyzed much faster than alkyl-α-glucosides. The enzyme acts on the maltose derivatives whose reducing groups have been masked. But among glucosylfructoses turanose, maltulose and isomaltulose were attacked with a slow rate while the enzyme was quite inert to sucrose. Malto- and isomalto-oligosaccharides were also hydrolyzed and the enzyme ceased its action at seven to eight units of hexose in both series of oligosaccharides.

The opt. pH range of Takamaltase was 4.2~4.6 and opt. temp., 50~55°C. Cu⁺⁺ and Hg⁺⁺ strongly inhibited the enzyme activity but other metal ions tested had no effects. It is suggested that the enzyme is not a sulfhydryl enzyme because of the lack of effects of SH-reagents on the activity.     

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

Summary The histo- and cytochemical localization of Ca⁺⁺-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca⁺⁺-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca⁺⁺ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg⁺⁺ in different concentrations were substituted for Ca⁺⁺ in the incubation medium the reaction was always reduced. Both Ca⁺⁺ and Mg⁺⁺ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity is discussed in relation to the regulation of the extracellular Ca⁺⁺ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Metal ion stimulators of PDE5 cause similar conformational changes in the enzyme as does cGMP or sildenafil

Purified PDE5 preparations exhibited variable proportions of two mobility forms (Bands 2 and 3) by native PAGE. Treatment of recombinant or native PDE5 with either cGMP or a substrate analog such as sildenafil, each of which is known to produce stimulatory effects on enzyme functions, caused a similar native PAGE band-shift to the lower mobility form (shift of Band 2 to Band 3). Incubation of PDE5 with Mg⁺⁺ or Mn⁺⁺, which is known to stimulate activity, caused a similar shift of the enzyme from Band 2 to Band 3 as did cGMP or sildenafil, but incubation with EDTA caused a time- and concentration-dependent shift to higher mobility (shift of Bands 2 and 3 to Band 1). A slow time course of the EDTA-induced band-shift suggested removal of a pre-bound metal ion (Me⁺⁺) with affinity of ~ 0.1 nM, which was similar to the previously determined affinity of PDE5 for Zn⁺⁺. The EDTA-treated enzyme (Band 1) could be shifted to Bands 2 and 3 by addition of cGMP, sildenafil, or Me⁺⁺; however, the cGMP- or sildenafil-induced shift was inhibited and the Me⁺⁺-induced shift was facilitated by treatment with EDTA. Results suggested that Me⁺⁺ removal from PDE5 produces a unique apoenzyme form (Band 1, more globular, negatively charged, or both) of PDE5 that can be partially converted to forms (Band 2, less globular or negatively charged, or both; and Band 3, more elongated/positively charged, or both) by addition of Me⁺⁺, substrate, or substrate analog. It is concluded that Me⁺⁺ causes conversion of PDE5 to similar conformational forms as caused by substrate or inhibitor binding to the catalytic site.     

Affinity Chromatographic Purification of β-Glucosidase of Candida Guilliermondii

Abstract

A 8-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethylcneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacryl-amide disc electrophoresis of the purified enzyme gave a single band. A K_m of 1.25 × 10^?4M was obtained using p_-nitrophenyl-β-D_-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at a temperature of 37°C. Enzyme activity was completely inhibited by Hg⁺⁺, Pb⁺⁺, and Zn⁺⁺ ions. The molecular weight of the enzyme is 48, 000 as estimated by sucrose density gradient centri-fugation.     

Enzyme activity and bacteriophage infection. I. Breakdown of adenosinetriphosphate

Experiments have been performed on the apyrase activity of E. coli, strain B. Although the dependence on pH and substrate is similar to that of rat tissue, the bacterial extracts are inhibited by Ca⁺⁺ and stimulated by Mg⁺⁺. In bacterial extracts the rate of phosphate release decreases in the course of the reaction, possibly owing to product inhibition. With multiple bacteriophage infection, the apyrase activity of the intact cells increased several fold, and the activity of extracts increased about 30 per cent. It is suggested that the changes could be attributed to an increase in the amount of enzyme although other alternatives cannot be precluded at present.     

Comparative Biochemical Studies of α-Glucosidases

The properties of brewer’s yeast α-glucosidase have been investigated. The enzyme was capable of hydrolyzing various α-glucosides and was active especially on aryl-α-glucosides in comparison with other α-glucosides and sugars. The rate of hydrolysis decreased in following order: phenyl-α-glucosides, sucrose, matlose and isomaltose.

The range of opt. temp, was 40~45°C and opt. pH, 6.5~7.0.

Cu⁺⁺ and Hg⁺⁺ inhibited strongly the enzyme activity and Zn⁺⁺, moderately. The enzyme was suggested to be a sulfhydryl enzyme from the inhibition experiments by SH-reagents and the effects of glutathione on the activity.

The enzyme synthesized some oligosaccharides from maltose. As the transglucosidation products, nigerose, isomaltose, kojibiose and maltotriose were detected by paperchromatography.

Pure nigerose was separated by splitting maltose with amyloglucosidase from the mixture of maltose and nigerose and by use of successive carbon column chromatography.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Ecto-ATPase

Summary An ecto-adenosine triphosphatase (E.C. 3.6.1.4 ATP-phosphohydrolase) is shown to be localised on the outer surface of varieties of cell membrane. The enzyme is different from the ATPase involved in biological energy transduction and ion transport mechanism. The characteristic of the enzyme lies in having a very broad substrate specificity and is inhibited by EDTA and higher concentration of ATP. The enzyme is dependent on bivalent metal ions, Mg⁺⁺ or Ca⁺⁺ for its optimum activity. The enzyme is highly sensitive to SH-reagents but insensitive to inhibitors of mitochondrial ATPase or Na⁺−K⁺-ATPase. The possible functions of the enzyme in being oriented outside the cell membrane is discussed.     

Activation of tubulinyl-tyrosine carboxypeptidase by spermine,spermidine and putrescine

Spermine, spermidine and putrescine produce dose dependent stimulation of the invitro tubulinyl-tyrosine carboxypeptidase. Maximal stimulation was obtained with spermine, spermidine or putrescine at 0.06 mM, 1 mM and 6 mM, respectively. At higher concentrations, the enzyme activity was inhibited. The enzyme was also activated by Mg⁺⁺; the concentration formaximal effect was 4–6 mM. The stimulation produced by optimal concentration of each amine was unaffected by Mg⁺⁺ up to 2 mM; higher concentration of Mg⁺⁺ showed inhibitory effect. At optimal Mg⁺⁺ concentration, the carboxypeptidase activity was inhibited by increasing amine concentration. The amines at 0.5 or 5 mM did not produce any effect on the incorporation of tyrosine catalyzed by tubulin tyrosine ligase.     

Studies on the Acid-protease of Paecilomyces varioti Bainier TPR-220

The crystalline acid-protease of Paecilomyces varioti Bainier TPR-220 is most active toward casein as substrate, at pH 3.0 and 60°C, and stable at pH 3.0 to 6.0 below 40°C. The enzyme decomposes protein molecules into smaller fragments than pepsin does and is inhibited by p-chloromercuri-benzoate, monoiodoacetate, sodium lauryl sulfate, iodine, potassium permanganate, N-bromosuccinimide, bacitracin, nitrofurylacrylamide, and Hg⁺ ion, but affected neither by metal ion except Hg⁺ ion, nor metal chelating agent, soy bean trypsin inhibitor, potato-protease inhibitior, cysteine, diiso-propylfluorophosphate, cyanogen bromide, and heparin. The presence of Ca⁺⁺, Co⁺⁺, Cu⁺⁺, Mg⁺⁺, Sr⁺⁺, and Zn⁺⁺ ions prevents heat inactivation of the enzyme.     

Studies in the enzyme make-up of Alternaria VIII. Pectic enzymes

Summary Present studies indicate that all the three species ofAlternaria possessed both intra- and extracellular PME activity but only the latter was significant. The role of the enzyme in pathogenicity of the strains has been discussed. The various optima determined for the extracellular enzyme activity were pH 4.5–7.0, temperature 50° C, time 24 hrs, and substrate concentration 1% pectin; the activity was proportional to enzyme concentration. Of the chemicals used to characterise the properties of the enzyme Na₂HPO₄, NaH₂PO₄, Mg⁺⁺, Ca⁺⁺ produced marked activation.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Studies on Phosphoprotein Phosphatase in Chick Embryo

Several properties of partially purified phosphoprotein phosphatase from chick embryo are described. The enzyme was active toward casein, phosvitin and phosphopeptone, but not toward low molecular weight phosphate esters including aliphatic and aromatic phos-phomonoesters, a phosphodiester, pyrophosphates and phosphoamides. The enzyme was not activated by reducing compounds. Heavy metal ions and sulfhydryl inhibitors inhibited the enzyme activity, but the inhibited enzyme was partially reactivated with cysteine. Metal chelating agents also inhibited the activity. To the oxalate treated enzyme, Fe⁺⁺ and Co⁺⁺ had a stimulatory effect. Differences in property between phosphoprotein phosphatases of chick embryo and of mammalian tissues are discussed.     

Studies on Isomerization of Sugars by Bacteria

An enzyme, which catalyzes the isomerization of d-glucose to d-fructose, has been found in a newly isolated bacterium which tentatively identified as Pacacolobacterum aerogenoides. The enzyme converts not only d-glucose but also d-mannose to d-fructose, and NAD and Mg⁺⁺ are required as cofactor for this isomerization. The properties of this enzyme were summarized as follows: (1) As a cofactor for the isomerization by this enzyme, NAD was absolutely necessary, whereas NADP, FMN and FAD were not. (2) The optimum pH was found to be at 7.5 and optinum temperature was at about 40°C. (3) The enzyme activity was markedly reduced by EDTA treatment and the reduced activity by EDTA was restored by the addition of Mg⁺⁺, Mn⁺⁺ or Co⁺⁺. (4) The enzyme activity was strongly inhibited by monoiodoacetate, p-chloromercuribenzoate, and Cu⁺⁺, however, the activity was recovered by adding cysteine or glutathione.     

Studies on Metabolic Pathway of NAD in Yeast

Quantitative studies on yeast 5′-nucIeotidase are presented.

K_m values for purine 5′-nucleotides were generally smaller than those for pyrimidine 5′-nucleotides and, among purine series, K_m value for 5′-AMP was the smallest, while their V values were almost same.

The enzyme activity was inhibited in the competitive type by bases, nucleosides, 3′- or 2′-nucleotides, and NMN and in the mixed type by NAD and NADP.

Base-, ribose-, 3′- or 5′-phosphate moiety of nucleoside and nucleotide had some effects on binding with enzyme; especially the structure of base moiety characterizes the K_m or K_i value.

The enzyme activity was accelerated by Ni⁺⁺ or Co⁺⁺, which increases V value but never affects K_m value.

The relationship between the structure of substrate and its affinity towards enzyme is discussed.     

Purification and properties of an NADP+-specific isocitrate dehydrogenase from Rhizobium meliloti

An NADP⁺-specific isocitrate dehydrogenase has been purified and characterized from Rhizobium meliloti. The enzyme showed Mn⁺⁺ or Mg⁺⁺ requirement. The apparent K_m values were 2.00×10^-5 m and 1.51×10^-5 m for dl-isocitrate and NADP⁺, respectively. The enzyme was inhibited by ATP, to a lesser extent by ADP and AMP. -Ketoglutarate also inhibited the enzyme activity. Oxalacetate and glyoxylate together inhibited the enzyme activity. The inhibition was competitive. Studies with thiol inhibitors suggested that the enzyme contained a sulfhydryl group at or near the active site. The enzyme has an approximate molecular weight of 60 000. Fluorescence studies suggested that the enzyme contained tryptophan     

Mn++-specific reactivation of EDTA inactivated alpha-isopropylmalate synthase from Alcaligenes eutrophus H 16.

The α-isopropylmalate synthase (EC 4.1.3.12) from $\text{Alcaligenes}\text{eutrophus}\text{H 16}$ was inactivated by EDTA in a time-dependent reaction. Only the addition of Mn⁺⁺ plus dithiothreitol could restore the activity. The substrate, α-ketoisovalerate, prevented the inactivation; the feedback inhibitor, leucine, and it's antagonist, valine, increased the rate of inactivation. Except for α,α′-bipyridyl, chelating reagents, other than EDTA had no effect on the enzyme stability. It is suggested that the α-isopropylmalate synthase is a metallo enzyme - the evidence points to Mn⁺⁺ as the metal ion - and that this enzyme uses a mechanism of catalysis which differs from that of the analogous malate synthase (EC 4.1.3.2) and citrate synthase (EC 4.1.3.4).     

The Characteristics of Arylamine N-Acetyltransferase in Pseudomonas aeruginosa

N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate. Characteristics of this bacterial NAT were further investigated. The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, the apparent K _m and V _max for 2-AF increased. The K _m and V _max were 0.504 ± 0.056 mM and 31.92 ± 3.23 nmol/min/mg protein, respectively, for the acetylation of 2-AF with 0.5 mM AcCoA. The optimum pH for the enzyme activity was estimated to be around 8.5. It was active at a temperature range from 5°C to 55°C, with maximum activity at 37°C. The enzyme activity was inhibited by divalent metal ions including Cu⁺⁺, Fe⁺⁺, Zn⁺⁺, Ca⁺⁺, Co⁺⁺, Mn⁺⁺, and Mg⁺⁺, suggesting that a sulfhydryl group is involved in the N-acetylation activity. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperature-dependent inhibition effect. Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, and the first two are likely to reside on the AcCoA binding site, but arginine residue may be located only near the AcCoA binding site. Our data demonstrate that P. aeruginosa possesses highly active N-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species. Received: 29 August 1997 / Accepted: 23 December 1997