Essential tyrosyl residues of human placental alkaline phosphatase

• 1.1. Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process.
• 2.2. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine.
• 3.3. The inactivation process showed saturation kinetics.
• 4.4. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate.
• 5.5. The binding affinity between the modified enzyme and inorganic phosphate was decreased.
• 6.6. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

     

Mitochondrial malic enzyme from the crayfish abdomen muscle,purification, regulatory properties and possible physiological role

• 1.1. Mitochondrial malic enzyme (l-Malate: NADP oxidoreductase (oxaloacetate decarboxylating) EC 1.1.1.40) has been isolated from abdomen muscle of crayfish Orconectes limosus by chromatography on Sepharose 6B and DEAE cellulose. Specific activity of the purified enzyme was about 5 μmols per min per mg protein, which corresponds to about 30-fold purification.
• 2.2. This enzyme showed extremely small reversiblity, since the reaction in the direction of decarboxylation is at least 37, 190 and 760 times that for the carboxylation at pH 7.0, 7.5 and 8.0 respectively.
• 3.3. Purified enzyme showed allosteric properties, which was more accentuated at more alkaline pH (Hill coefficients were 1.1, 1.7 and 1.8 at pH 7.0, 7.5 and 8.0 respectively). The activity of malic enzyme was increased considerably in the presence of succinate and fumarate.
• 4.4. Mitochondira isolated from abdomen muscle of Orconectes limosus incubated in the presence of malate, fumate and succinate catalysed pyruvate production which was stimulated by ADP and inhibited by respiratory chain inhibitors.
• 5.5. NADH but not NADPH oxidation was catalysed by broken mitochondria or sonic particles. When NADPH and NAD were added simultaneously the rate of oxidation. This suggests the presence of active NADPH:NAD transhydrogenase in mitochondria isolated from the crayfish abdomen muscle.
• 6.6. A possible metabolic role for NADP-linked malic enzyme/transhydrogenase couple in abdomen muscle of crayfish Orconectes limosus is proposed.

     

The effect of γ-radiation on the NADP-MALIC enzyme from mango fruit,mangifera indica—I. Physico-chemical aspects

• 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in dilute solution and the effect of γ-irradiation was investigated.
• 2.2. The activity of the enzyme decreased exponentially as a function of the applied dose under all conditions investigated. The inactivation yield (Go-value) in neutral solution and in air was 0.069.
• 3.3. The role of the radicals produced by water radiolysis in the inactivation of the enzyme was investigated by using different gas atmospheres and selective free radical-anions. The hydrogen atom and the hydrated electron (reducing species) were found to be important in the enzyme inactivation; as well as the possible destruction of cysteine and tryptophan residues.
• 4.4. The irradiated enzyme appears to adopt a more compact conformation as reflected in a slightly lower M_r, Stokes-radius and diffusion coefficient.
• 5.5. γ-Radiation does not lead to any heterogeneity in the charge and size properties of the enzyme and the pI and the M_r of the subunits were unaffected.
• 6.6. Some differences in the amino acid composition of the non-irradiated and irradiated enzyme were observed but specific amino acid residues were not preferentially destroyed.
• 7.7. These changes were also reflected in the ultraviolet spectrum of the enzyme which shifted to lower values.
• 8.8. The major cause of inactivation seem to be a change in conformation caused by chemical modification of amino acid side chains.

     

Regulation of horse-liver glutathione reductase

• 1.1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations.
• 2.2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective.
• 3.3. GSSG fully reactivated the inactive enzyme at 38°C and neutral to acidic pH (5.5–7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.

     

In vitro non-enzymatic glycosylation of myofibrillar proteins

• 1.1. Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered.
• 2.2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered.
• 3.3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated.
• 4.4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation.
• 5.5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.

     

Kinetic analysis of reversible closed bicyclic enzyme cascades covering the whole course of the reaction

A kinetic analysis of the closed bicyclic enzyme cascades is presented.

• 1.1. It includes the dependence on time from the onset of the reaction, of the concentration of the modified and unmodified enzyme species involved and the time course equations of the modificational fractions of the interconvertible enzymes.
• 2.2. The transient phase equations obtained allow the definition of new regulatory modification properties.
• 3.3. The expressions for concentrations of the unmodified and modified forms of the interconvertible enzymes, as well as those of the fractional modifications in the steady state are derived as particular cases of the general equations.
• 4.4. These steady state expressions coincide with those obtained by other authors.
• 5.5. The analytical results obtained are discussed in relation to the Escherichia coli glutamine syntethase cascade.

     

Decrease in yeast glucose-6-phosphate dehydrogenase activity due to oxygen free radicals

• 1.1. u.v. radiations and copper acetate, as free radical generating systems, determine a significant diminishing of glucose-6-phosphate dehydrogenase activity in the homogenates of Saccharomyces cerevisiae.
• 2.2. The inactivation is proportional to the concentration of the formed free radicals, existing a direct dependence on the action time of the free radicals generating systems and on the irradiation dose. The decrease of the enzyme catalytic activity is correlated with the increase of the malondialdehyde concentration.
• 3.3. The affinity for the substrate of the enzyme under the action of free radicals does not change significantly compared to the native enzyme: the K_m value for NADP is halved, whilst that for glucose-6-phosphate remains unchanged.
• 4.4. The electrophoretic study shows evidence of five electrophoretic bands with enzymatic activity in the native extract and the disappearance of one molecular form under the free radical action.

     

NADPH/NADP ratio could regulate the glyoxylate cycle in Tetrahymena pyriformis

• 1.1. The glyoxylic acid cycle pathway could be regulated through the modulation of the isocitrate dehydrogenase-NADP activity. This enzyme is inhibited by NADPH.
• 2.2. The effect on the glyoxylate cycle flux of variations in the rate of the NADPH-consuming pathways has been studied.
• 3.3. Increase in the rate of NADPH-consuming activity by addition of H₂O₂ produces inhibition of the glyoxylate cycle and decrease in the NADPH/NADP ratio.
• 4.4. These results suggest that the glyoxylate flux in Tetrahymena could be modulated by regulation of NADP-dependent isocitrate dehydrogenase by the NADPH/NADP ratio.

     

Studies on the active centre(s) of rat liver porphyrinogen carboxy-lyase. in vivo effect of hexachlorobenzene on decarboxylation site(s) of porphyrinogens

• 1.1. The role of histidine on the decarboxylation of porphyrinogens of 7-, 6-, and 5-COOH III brought about by porphyrinogen carboxy-lyase (PCL) was studied.
• 2.2. For this purpose hepatic PCL from normal and hexachlorobenzene (HCB) treated rats were modified with diethylpyrocarbonate.
• 3.3. The results indicated that the enzyme from both normal and porphyric animals had histidine at the binding sites of all the porphyrinogens assayed.
• 4.4. Comparative studies between the enzyme from normal and porphyric rats suggested that in vivo HCB treatment affected the active site for the decarboxylation of 7-, 6- and 5-COOH porphyrinogens III at histidine residues.
• 5.5. On the other hand arginine modification by 2,3-butanedione treatment altered 5-COOH porphyrinogen III decarboxylation for both enzymes. However this amino acid was not involved at the binding site of this substrate.

     

Effects of high dietary methionine on activities of selected enzymes in the liver of kittens (felis domesticus)

• 1.1. Growing male kittens were fed an 18% casein diet supplemented with 2, 3, or 4% l-methionine (MET) for 6 weeks.
• 2.2. Free MET concentration in liver increased 30-fold and cystathionine two- to three-fold; the activity of adenosyl-MET transferase and cystathionase also increased but remained lower than previously found in rats.
• 3.3. Taurine concentration in liver decreased in cats fed excess MET and appeared to depend on taurine intake.
• 4.4. Alanine aminotransferase activity was high in all groups while serine dehydratase activity was very low.
• 5.5. Pyruvate kinase and malic enzyme activities which are normally low in cat liver increased after excess MET. Also, glucose 6-phosphate and 6-phosphogluconate dehydrogenases increased.
• 6.6. Cat liver metabolism showed limited adaptation to an excess dietary intake of methionine compared to that found in rats.

     

On the synthesis and incorporation of catalase and urate oxidase into the peroxisomes of mouse liver

• 1.1. The processes associated with the biogenesis of peroxisomes in mouse liver have been studied by following the incorporation of radiolabelled leucine into major enzymic components of this organelle.
• 2.2. Maximal incorporation of label into peroxisomal catalase and urate oxidase occurred within 2 hr, with the urate oxidase being labelled before catalase, but subsequent to the incorporation of phospholipid into this organelle.
• 3.3. Subsequently, immunoprecipitation of catalase from the large granular fraction of mouse liver was shown to result in the isolation of a catalase molecule which had lost a peptide of approx. 2000 dalton from each subunit by comparison with the newly-synthesized enzyme.
• 4.4. It was observed that the modification of catalase was obviated by the presence of leupeptin and iodoacetamide and this information has enabled the purification of both modified and unmodified forms of the enzyme.
• 5.5. The possible significance of these data has been discussed and the major features incorporated into a working model of peroxisomal biogenesis.

     

Purification and characterization of hatching enzyme of the pike (Esox lucius)

• 1.1. A choriolytic enzyme was isolated from the hatching medium of the pike, Esox lucius.
• 2.2. The enzyme is defined as hatching enzyme.
• 3.3. The molecular weight of the enzyme is 24,000.
• 4.4. The enzyme is a glycoprotein containing 2% carbohydrate.
• 5.5. Its isoelectric point is 6.5.
• 6.6. The pH optimum is around pH 8.
• 7.7. The enzyme molecule contains two disulfide bonds but no free cysteine.
• 8.8. Inhibitor studies and metal analysis show that the enzyme is a zinc-metalloprotease.

     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

     

Selective modification of tryptophan-150 in ovine placental lactogen

• 1.1. Ovine placental lactogen was modified by reaction with o-nitrophenylsulfenyl chloride. Fluorescence measurements indicated that one of the two tryptophan residues of the molecule had reacted. Besides, there was some reagent not covalently bound.
• 2.2. The reagent was covalently bound to Trp-150. No evidence of modification of Trp-90 was found.
• 3.3. Binding capacity to lactogenic as well as somatogenic receptors was diminished but not abolished upon modification, indicating that absolute molecular integrity of Trp-150 is not required for binding.
• 4.4. This behavior is similar to that of the tryptophan residues of ovine prolactin.

     

Identification of essential amino acids in the active center of thyroidal NAD+ glycohydrolase

• 1.1. Purified thyroidal NAD⁺ glycohydrolase has been subjected to the action of a number of group specific reagents in order to gain information concerning its mode of action.
• 2.2. Modification of histidyl residues with diethylpyrocarbonate strongly suppresses the NAD⁺ glycohydrolase activity. Inactivation with this reagent can be reversed to some extent by subsequent treatment with hydroxylamine.
• 3.3. NAD⁺ and ADP-ribose partially protect against inactivation with similar efficiencies.
• 4.4. The incomplete reactivation with hydroxylamine after diethylpyrocarbonate treatment and the selective inactivation by 2,4-pentanedione indicates that apart from one or more essential histidyl residue(s) also lysyl residues are important for activity. NAD⁺ and to a smaller extent ADP-ribose again protect against inactivation by 2,4-pentanedione.
• 5.5. The sensitivity of the enzyme towards N-ethyl-5-phenyl-isooxazolium-3'-sulfonate further points to the importance of carboxylate containing side chains.
• 6.6. The mechanistic implications of these results are discussed.

     

Phosphonomonoesterase activity in Metridium senile L.

• 1.1. An esterase which hydrolyzes 4-nitrophenyl(phenyl)phosphonic acid (4-NPPP) was purified from M. senile (sea anemone).
• 2.2. The enzyme showed no 5′-nucleotide phosphodiesterase activity with 5′-(4-nitrophenyl) TMP or phosphomonoesterase activity with 4-nitrophenylphosphate.
• 3.3. Addition of excess Zn²⁺ restored activity after inactivation by EDTA.
• 4.4. Thiol reagents and phenylmenthanesulfonylfluoride did not inactivate, whereas, dithiothreitol inactivated.
• 5.5. Aminoethylphosphonic acid (AEP) was a competitive inhibitor of 4-NPPP indicating possible activity with phosphonomonoesters of AEP.

     

The effect of prolonged fasting on total lipid synthesis and enzyme activities in the liver of the European eel (Anguilla anguilla)

• 1.1. The extent of fatty acid synthesis from [1-¹⁴C]acetate in liver slices was reduced 6-fold when eels were fasted for 1–7 weeks and 20-fold when fasted for 39 weeks; thereafter hepatic lipogenesis seemed to remain constant for up to 95 weeks of fasting.
• 2.2. After a 1–3 week fast some hepatic enzyme activities were reduced (acetyl-CoA carboxylase decreased 2-fold and fatty acid synthetase declined 5-fold), while others remained unchanged (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, α-glycerol phosphate dehydrogenase as well as malic enzyme and ATP-citrate lyase).
• 3.3. The optimum temperature for measuring both total lipid synthesis and lipogenic enzyme activity in eel liver was found to be 30°C.

     

Inactivation of ornithine decarboxylase by intermediates of tyrosinase-catalyzed reaction

• 1.1. Intermediates in the process of melanin synthesis formed through oxidation of catechols by tyrosinase produced the inactivation of ornithine decarboxylase (ODC), a key enzyme in the polyamine biosynthesis pathway.
• 2.2. The inactivation was dependent on the substrate used (dihydroxybenzylamine ⪢ l-3,4-dihydroxy-phenylalanine ⪢ l-tyrosine) and on the concentration of intermediate produced rather than on the rate of formation.
• 3.3. Sulfhydryl compounds (dithiothreitol and glutathione) or quinone-reducing agents (ascorbic acid) prevented the inactivation of ODC; l-ornithine, but not other aminoacids, also protected partially ODC. The results suggest that different cysteine residues in ODC molecule are implicated in the inactivatory event.
• 4.4. When ¹⁴C-labeled catechols were used, numerous polypeptides resulted labeled, showing that the reactive quinones formed as intermediates in the process of melanin biosynthesis bind covalently to many cellular proteins.

     

Partial purification and characterization of cysteine proteinase from metamorphosing tadpole tails of Rana catesbeiana

• 1.1. A leupeptin-sensitive proteinase was partially purified from regressing tadpole tails by acetone factionation and column chromatography on S-Sepharose.
• 2.2. The enzyme degraded hemoglobin and myoglobin at pH 3.0. The enzyme also hydrolyzed Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA at pH 4.0.
• 3.3. The enzyme activity was inhibited by leupeptin, egg cystatin, E-64 and monoiodoacetic acid and was activated by l-cysteine.
• 4.4. The enzyme degraded myosin and actin in myofibrils of tadpole tails.
• 5.5. The enzyme belongs to the cysteine proteinase and is possibly involved in tail degradation during the metamorphosis of tadpoles.

     

The anaerobic metabolism of malate of Saccharomyces bailii and the partial purification and characterization of malic enzyme

1. The main pathway of the anaerobic metabolism of l-malate in Saccharomyces bailii is catalyzed by a l-malic enzyme.
2. The enzyme was purified more than 300-fold. During the purification procedure fumarase and pyruvate decarboxylase were removed completely, and malate dehydrogenase and oxalacetate decarboxylase were removed to a very large extent.
3. Manganese ions are not required for the reaction of malic enzyme of Saccharomyces bailii, but the activity of the enzyme is increased by manganese.
4. The reaction of l-malic enzyme proceeds with the coenzymes NAD and (to a lesser extent) NADP.
5. The K _m-values of the malic enzyme of Saccharomyces bailii were 10 mM for l-malate and 0.1 mM for NAD.
6. A model based on the activity and substrate affinity of malic enzyme, the intracellular concentration of malate and phosphate, and its action on fumarase, is proposed to explain the complete anaerobic degradation of malate in Saccharomyces bailii as compared with the partial decomposition of malate in Saccharomyces cerevisiae.