Factors affecting the reassociation and reactivation of the half-molecular weight nonidentical subunits of pigeon liver fatty acid synthetase.

The pigeon liver fatty acid synthetase complex (14 S) is dissociated in low ionic strength buffer containing dithiothreitol to form a half-molecular weight subunits (9 S) which are completely inactive for the synthesis of saturated fatty acids. The dithiothreitol-protected (reduced) subunits are rapidly reassociated and reactivated to form the active enzyme complex, not only by an increase in salt concentration but also by micromolar concentrations of NADP+ or NADPH. Increases in KCl or NADPH concentration result in an increase in the extent of reactivation (equilibrium) with no change in the over-all rate of the reaction or the half-life ofreactivation of the enzyme. The extent (equilibrium) of reactivation of the enzyme is the same in 0.2 M potassium phosphate buffer, pH 7.0; 0.2 M KCl in 5 mM Tris-35 mM glycine buffer, PH 8.3; and 50 muM NADP+ or NADPH in the Tris-glycine buffer. The extent and rate of reactivation of the enzyme is dependent not only on ionic strength and NADPH concentration, but also on pH and temperature. Reactivation with 0.2 M KCl is optimal between pH 7.3 and 8.5. At higher and lower pH values the rate and extent of reactivation are lowered. The rate and extent of reactivation are also decreased as the temperature is lowered below 10 degrees. At 0 degrees there is little reactivation of enzyme activity. However, in the presence of 0.2 M KCl containing 15 to 40% glycerol at 0 degrees, reactivation of the enzyme is about 50% complete. The rate of reactivation of enzyme in the presence of KCl or NADPH conforms to first order kinetics. This result suggests that the subunits first combine to form an inactive complex which is subsequently transformed to an enzymatically active complex. Evidence for the presence of inactive complex was obtained in experiments carried out in 0.2 M KCl at pH 6.0, and in 0.2 M KCl at pH 8.3, at both 6 and 3 degrees. Under these conditions the amount of complex observed upon ultracentrifugation was greater than expected from determinations of enzyme activity. The above findings suggest that ionic and hydrophobic interactions, and possibly the water structure surrounding the interacting sites, are of prime importance in reassociation and reactivation of enzyme. In addition, NADP+ and NADPH have very specific effects in bringing about reassociation and in maintaining the structural integrity of the multienzyme complex.     

Some properties of a deoxyribonuclease isolated from Aspergillus niger

A deoxyribonuclease, isolated from the mycelia of Aspergillus niger, grown as surface cultures on a liquid medium, was partially purified and had an optimum pH of 9.5 and an optimum temperature of 37°C. The enzyme was strongly activated by Mg²⁺ while Mn²⁺, Ca²⁺ or Co²⁺ activated the enzyme to lesser extents. Thiol reagents, mercaptoethanol and dithiothreitol (DTT) activated the enzyme. S-Adenosylmethionine at low concentration (2.8?5.0 × 10^?2 mM) activated the enzyme but at a higher concentration (11.5 × 10^?2 mM) and above it inhibited the enzyme. The effect of EDTA on the enzyme was variable. The enzyme had both ATP-dependent and independent activities, the former usually being higher. ATP could be replaced by CTP or GTP. The nucleoside diphosphates ADP, CDP and GDP could replace ATP but they were not as effective as the triphosphates.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS : I. The Number of Acetylcholinesterase Molecules and of Other DFP-Reactive Sites at Motor Endplates, Measured by Radioautography

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-³²P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10⁷ sites per endplate, in the diaphragm, about 3 x 10⁷. Reaction with DFP-³²P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10⁷ and 1.1 x 10⁷per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.     

A cold-labile acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver. Reactivation and reconstitution of the active species from the inactive monomer

An acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold-induced inactivation and dissociation of the enzyme: At a protein concentration of 14 micrograms/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37 degrees C appeared to be optimal for reactivation; warming at 25 degrees C or at 43 degrees C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme-protein concentration of 14 micrograms/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl-CoA (0.5 mM) restored the activity about 50%. High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity. Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4 degrees C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.     

l-Phenylalanine stereospecifically inhibits the renaturation of muscle pyruvate kinase

Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine HCl. Subsequent dilution of the enzyme into buffer containing β-mercaptoethanol or dithiothreitol results in recovery of enzymatic activity with an average half-time of 17 min at 16 °C. The addition of 1 mm l-phenylalanine increases the average half-time for recovery of enzymatic activity to 26 min, while 8 mm l-phenylalanine further increases this value to 46 min. Tyrosine and tryptophan also inhibit the reactivation but to a lesser extent than phenylalanine. Neither l-alanine, l-valine, d-phenylalanine, phosphoenolpyruvate, nor fructose 1,6-bisphosphate have any appreciable effect on activity recovery rates, either in the presence or absence of l-phenylalanine. Phenylpyruvate is a very potent inhibitor of reactivation. The addition of 5 mm phenylpyruvate increases the half-time to 57 min. The evidence presented in this paper supports the hypothesis that an l-phenylalanine-binding site which probably is distinct from the catalytic site is formed early in the renaturation process. l-Phenylalanine binds to this site and inhibits two first-order relaxations that are rate limiting for the reactivation and that have the following rate constants: 8.76 × 10^?2 and 1.24 × 10^?2 min^?1, respectively, in the absence of phenylalanine and 3.04 × 10^?2 and 7.63 × 10^?3min^?1, respectively, in the presence of 8.0 mm phenylalanine. We presume these first-order processes to be transconformational steps in the reactivation process.     

Cold-lability of prolyl-tRNA synthetase from higher plants

Pro-tRNA synthetase from D. regia and P. aureus lost enzymic activity more rapidly at 0° than at room temperature. The enzyme from a number of higher plants that produce azetidine-2-carboxylic acid (A2C) was more rapidly inactivated in the cold than the enzyme from plants which do not contain A2C. The rate of cold inactivation was dependent on temperature and on the concentration of glycerol, protein and sulphydryl-reducing reagents. Substrates of Pro-tRNA synthetase also stabilized the enzyme against cold inactivation. Enzyme which had been completely inactivated by storage in the cold, could be reactivated by warming in the presence of a sulphydryl-reducing reagent. The rate of reactivation was dependent on temperature, pH and the concentration of sulphydryl-reducing reagent. Kinetic analysis indicated the existence of more than one molecular form of the enzyme. It is suggested that the cold-lability of Pro-tRNA synthetase may be due to dissociation of the active enzyme molecule into inactive subunits.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca²⁺ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH₃) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca²⁺ concentrations and inhibited by higher (μM range) concentrations of the cation. A 2–3-fold stimulation of the activity in response to Ca²⁺ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca²⁺ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca²⁺ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca²⁺ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca²⁺. Mg²⁺ concentrations in the incubation also influenced the Ca²⁺ concentration dependence of adenylate cyclase; at high Mg²⁺ more Ca²⁺ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH₃ cells only in the presence of Ca²⁺; as Ca²⁺ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca²⁺ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH₃ cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca²⁺. Increasing free Ca²⁺ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Structural and biochemical properties of lichenase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu²⁺ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu²⁺ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni²⁺, Co²⁺ and Zn²⁺ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)₈-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.     

SUBUNIT STRUCTURE AND KINETIC PROPERTIES OF 4-AMINOBUTYRATE-2-KETOGLUTARATE TRANSAMINASE PURIFIED FROM MOUSE BRAIN

Abstract— The effects of divalent metal ions, sulfhydryl reagents, carbonyl trapping reagents, substrate analogs, and organic solvents on purified mouse brain 4-aminobutyrate-2-ketoglutarate transaminase (EC 2.6.1.19) and the subunit structure of this enzyme were studied. Of the metal ions tested, Hg²⁺ was found to be the most potent inhibitor inhibiting the enzyme 50 percent at a concentration of 0-7 μM. The order of decreasing inhibitory potency for the divalent metal ions was: Hg²⁺± Cd²⁺± Zn²⁺± Cu²⁺± Co²⁺± Ba²⁺± Sr²⁺± Ni²⁺± Mn²⁺± Ca²⁺± Mg²⁺. p-Chloromercuribenzoale was the most potent inhibitor among the sulfhydryl reagents tested inhibiting the enzyme to the extent of 50 per cent at 0-5 μM 3-Mercaptopropionic acid was found to be a competitive inhibitor for GABA and non-competitive for 2-ketoglutarate. The K_i, value was estimated to be 13 μM. Aminooxyacetic acid was the most potent inhibitor of the carbonyl trapping agents with a K, value of 0-06 μM. being competitive with GABA and non-competitive with 2-ketoglutarate. Hydroxylamine and hydrazine were the next most potent compounds in this group. Of a series of substrate analogs and metabolites tested, only acetic acid, propionic acid, butyric acid, glutamic acid, adipic acid, pimelic acid and 2-ketoadipic acid inhibited the enzyme to a significant extent. Dioxan inhibited the enzyme 50 per cent at a concentration of 5 per cent (v/v) whereas methanol and ethanol only inhibited 5-10 per cent at 10 per cent (v/v) concentration. A spectrum of the native enzyme at pH 7-2 showed maxima at 278 nm. 330 nm and 411 nm. Treatment of the enzyme with aminooxyacetic acid or 3-mercaptopropionic acid caused the maximum at 411 nm to disappear. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed two protein bands. The molecular weights of these two subunits were determined to be 53.000 and 58,000, respectively.     

Effect of the lyotropic series of anions on denaturation and renaturation of 20β-hydroxysteroid dehydrogenase

The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20β-hydroxysteroid dehydrogenase (17,20β,21-trihydroxysteroid:NAD⁺ oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denaturated enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the inactivation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1–2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.     

Purification and Properties of Gitrus natsudaidai Pectinesterase

The level of glutamine synthetase in Micrococcus glutamicus ATCC 13032 varied in response to the nitrogen source in culture medium; it was 10?20 fold higher in glutamate-, peptone- or yeast extract-grown cells than in ammonia- or urea-grown cells. Ammonia (3 mM) reduced the enzyme level to 50% when added to glutamate medium. No difference between nitrogen sources was observed in extent of inhibition by Mg²⁺ of γ-glutamylhydroxamate-forming (transferring) reaction in crude extracts.

The optimum pH was 7.0 ? 8.0 for glutamine-forming (synthesizing) reaction and 7.0 for transferring reaction. The enzyme was stable to heating at 50°C for 10 min in 0.05 M potassium phosphate buffer (pH 6.0) containing 0.1 mM MnCl₂. Km values for glutamate, ammonia and ATP in synthesizing reaction were 7.9, 5.0 and 1.2 mM, respectively. GTP and hydroxylamine could be substituted for ATP and ammonia with about 10 and 30% reactivity. Mg²⁺ was effective as a cofactor in synthesizing reaction and Mn²⁺ showed 34% of the reactivity of Mg²⁺ at a concentration of 30 mM. Glutamine synthetase was inhibited by adenosine, AMP and ADP but not by amino acids other than D-threonine. The regulation system of glutamine synthetase in M. glutamicus is discussed.     

Studies on the phosphomannose isomerase of Amorphophallus konjac C. Koch II. Effect of divalent metal ions on the EDTA-treated enzyme

Konjak phosphomannose isomerase was inactivated in a time-dependentprocess by metal binding agents, and the inactivated enzymewas instantaneously reactivated by adding such metal ions asZn⁺⁺, Co⁺⁺, Fe⁺⁺, Mn⁺⁺ and Cu⁺⁺. However, neither Ca⁺⁺ nor Mg⁺⁺were effective for reactivation. Zn⁺⁺, at a low concentration,brought about complete reactivation of the enzyme at pH 6–7. The EDTA-treated enzyme was more susceptible to heat denaturationwhen compared with the native enzyme, but the addition of variousmetal ions caused the recovery of the thermal stability of theEDTA-treated enzyme. The magnitude of the recovery dependedon the metal ion species and the concentrations. The most effectivemetal ion was Co⁺⁺, which caused the recovery of thermal stabilityto a level higher than that of the native enzyme. Phosphomannoseisomerase was inhibited by pchloromercuribenzoate and HgCl₂;the inhibition by p-chloromercuribenzoate being more pronouncedas incubation progressed. In contrast, the EDTA-treated enzymewas more readily inhibited by the mercurial ion than was thenative enzyme. Zn⁺⁺, when added to the EDTA-treated enzyme,markedly restored its resistance to the mercurial-induced inhibition.The metal-substituted enzyme was also inhibited by EDTA in atime-dependent process. ¹ This paper constitutes part 4 of studies on konjak mannanbiosynthesis. (Received March 3, 1975; )     

Inhibition of muscle phosphorylase a by natural components of the sarcoplasm

Using a reconstituted glycolytic enzyme system from muscle tissue, it was shown that phosphorylase activity was regulated by some process to provide only the required amount of glucose 1-phosphate, regardless of the percentage of phosphorylase in the a form. By carrying out phosphorylase a assays at high enzyme concentration (2 mg ml^?1), the same concentration as in the reconstituted system and comparable with in vivo, it was shown that (a) the K_m for phosphate was higher and V lower than at low enzyme concentration (2 μg ml^?1), (b) the presence of other glycolytic enzymes at 40 mg ml^?1 suppressed the activity a further threefold, and (c) phosphocreatine inhibited the enzyme. Taken together, these three effects were sufficient to explain the relative lack of activity of phosphorylase a in the reconstituted system. The inhibition by phosphocreatine is seen as a mode of feedback control on phosphorylase activity in vivo.     

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.     

Effect of detergents on the enzyme activities of the rat liver plasma membrane

The anionic detergents sodium dodecyl sulfate (SDS) and Alipal CO-433 and the non-ionic detergent Trition X-100 at concentrations of 0.02–0.10% cause a more rapid solubilization of phospholipid than proteins in isolated rat liver plasma membranes. All three detergents cause an increase in membrane turbidity at low detergent concentration (0.01–0.04%) but then decrease the turbidity at higher detergent concentration (0.04–0.10%). Each detergent gives a characteristic turbidity-detergent concentration profile which is pH dependent.The activities of the membrane-bound enzymes Mg²⁺ ATPase, 5′-nucleotidase and acid and aklaline phosphatase were influenced by each detergent to a different extent. Each enzyme gave a characteristic activity-detergent concentration profile. Mg²⁺ ATPase was inhibited by all detergents. 5′-Nucleotidase was stimulated by Triton and Alipal but inhibited by SDS. Alkaline phosphatase was stimulated by Alipal and SDS and not influenced by Triton. Acid phosphatase was stimulated by Triton and inhibited by Alipal and SDS. 56% of the total membrane-bound alkaline phosphatase and 23% of the total membrane-bound 5′-nucleotidase was solubilized in an active form by 0.06% and 0.05% SDS respectively.     

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo: Control by High CO2 Concentration

High CO₂ concentrations (HC) in air induce partial deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39). Under saturating irradiance, increase in [CO₂] to 1 200 cm³ m^–3 reduces the concentration of operating carboxylation centres by 20–30 %. At a further increase in [CO₂], the activity remained on the same level. Under limiting irradiance, the lowest activity was reached at 600 cm³(CO₂) m^–3. The presence of oxygen diminished deactivation, but O₂ failed to stimulate reactivation under high CO₂. Conditions that favour oxygenation of ribulose-1,5-bisphosphate (RuBP) facilitated reactivation. Even HC did not act as an inhibitor. HC induces deactivation of RuBPCO by increasing the concentration of free reaction centres devoid of the substrate, which are more vulnerable to inhibition than the centres filled with substrates or products.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

Studies on nucleotidases in plants. Reversible denaturation of the crystalline mung bean nucleotide pyrophosphatase and the effect of adenylates on the native and renatured enzyme

The crystalline mung bean nucleotide pyrophosphatase was inhibited nonlinearly by AMP, one of the products of the reaction. The partially inactive enzyme was specifically reactivated by ADP, and V at maximal activation was the same as that of the native enzyme. ATP was a linear, noncompetitive inhibitor. The kinetic evidence suggested that ADP and ATP might not be reacting at the same site as AMP. The electrophoretic mobility of the enzyme was increased by AMP, whereas ADP and ATP were without effect.The enzyme was denatured on treatment with urea or guanidine hydrochloride. The renatured and the native enzyme had the same pH (9.4) and temperature (49 °C) optimum. The K_m (0.2 mm) and V (3.2) of the native enzyme increased on renaturation to 1.8 mm and 8.0, respectively. In addition, renaturation resulted in desensitization of the enzyme to inhibition by low concentrations of AMP. Renaturation did not affect the reactivation of the apoenzyme by Zn²⁺.