Long-chain saturated fatty acids can be α-oxidised by a purified enzyme (Mr 240 000) in cucumber (Cucumis sativus)

An enzyme (M_r 240 000) with high fatty acid α-oxidation activity has been purified from the fruit of cucumber (Cucumis sativus). The specific α-oxidation activity in the purified fraction was 370 nmol/min per mg protein determined as liberation of ¹⁴CO₂ from [1-¹⁴C]palmitic acid. α-Oxidation activity was observed both in the 12 000×g pellet and 150 000×g pellet by differential fractionation of cucumber homogenate. The enzyme was purified about 220-fold to near homogeneity from a 12 000×g fraction by solubilisation with Triton X-100R, ammonium sulphate precipitation, hydrophobic interaction and anion-exchange chromatographies and Superose 12 gel filtration. The molecular mass of the native enzyme was 240 000, and the major subunit molecular mass of 40 000 indicated an oligomeric structure.     

Calpain activity alters in rat myocardial subfractions after ischemia or reperfusion

To examine whether calpain is activated during ischemic or reperfusion injury, we measured calpain activity of the subfractions of rat myocardia after global ischemia for 60 min or the ischemia followed by 30 min reperfusion by the Langendorff procedure. The myocardial homogenate was fractionated into 600 × g, 10 000 × g and 100 000 × g pellet fractions as well as 10 000 × g supernatant fraction. The supernatant fraction was further subjected to DEAE cellulose and phenyl-Sepharose chromatographies to separate μ- and m-calpains. The m-calpain activity of the DEAE fractions after global ischemia for 60 min was higher but that after ischemia-reperfusion was lower than that of the control. On the other hand, the ischemia-reperfusion but not ischemia by itself raised the calpain activity of the phenyl-Sepharose fraction (μ-calpain) and the 10 000 × g pellet measured at 100 μM and 5 mM Ca²⁺. Treatment with verapamil but not with ryanodine during ischemia attenuated the increase in m-calpain activity. A dot-blotting analysis of calpain antigenicity showed a decrease in soluble but no change in the particulate fractions after ischemia-reperfusion. An immunoblotting technique did not detect proteolysis of the calpain 80-kDa subunit. These observations suggest that calpain is activated by Ca²⁺ influx during ischemia and reperfusion without gross changes in its amount. Some unknown processes other than translocation or autolysis are thought to be involved in the alterations.     

High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Aryl hydrocarbon hydroxylase activity in the fungus Cunninghamella bainieri: evidence for the presence of cytochrome P-450.

The aryl hydrocarbon hydroxylase (AHH) enzyme from the fungus Cunninghamella bainieri has been characterized. It is NADPH dependent and exhibits a pH optimum near 7.8. It is inhibited by CO, SKF 525-A, and metyrapone, but cyanide shows no inhibitory effect. These data, together with the pattern of inhibition and stimulation shown by metal ions, suggest that the fungal AHH activity is due to a cytochrome P-450. About 25% of the hydroxylase activity remains in the supernatant while the remainder precipitates after centrifugation at 100,00g for 2.5 h. The 100,000g supernatant was further fractionated by (NH₄)₂SO₄ precipitation. A NADPH-dependent cytochrome c reductase is concentrated mainly in the 100,000g supernatant, and a cytochrome c oxidase is present mainly in the 100,000g pellet. The cytochrome c reductase is essential for AHH activity as shown by the inhibition of AHH activity with cytochrome c and dichloroindophenol. Solubilization of a portion of the 100,000g pellet in aqueous digitonin followed by dithionite reduction and addition of CO resulted in the observation of a maximum absorbance at 450 nm characteristic of cytochrome P-450.     

Partial purification and characterization of 3-hydroxy-3-methylglutaryl coenzyme a reductase from the leaves of guayule (Parthenium argentatum)

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been isolated and was partially purified from the leaves of Parthenium argentatum. The enzyme was found to be associated both with the cytosol and the chloroplasts. Ten mM dithiothreitol was essential to prevent loss of activity. Optimum activities of cytosolic and chloroplastic fractions were observed at pH 7.0 and 7.5 respectively. Preincubation of the reaction mixtures with CoA, acetyl-CoA, σ-phenanthroline and iodoacetamide resulted in the progressive loss of enzyme activity. 3-Hydroxybutyrate and mevalonate also inhibited the enzyme. The Michaelis constants of the enzyme for HMG-CoA and NADPH were 0.25 and 0.31 mM respectively for the cytosolic enzyme, while those for the chloroplastic enzyme were 0.018 and 0.42 mM respectively. Inhibition studies indicated that hydroxybutyrate was a competitive inhibitor with respect to HMG-CoA. The inhibition of mevalonate was competitive with HMG-CoA and non-competitive with NADPH.     

Properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pisum sativum seedlings.

The properties of 3-hydroxy-3-methylglutaryl coenzyme A reductase from the microsomal fraction of Pisum sativum seedlings have been described. The enzyme requires NADPH for activity and NADH does not support the reaction. The presence of a thiol compound such as dithiothreitol, is required for activity and a concentration of 10 mmm is optimal. The pH optimum is 6.8 and the K_m (apparent) for dl-3-hydroxy-3-methylglutaryl coenzyme A is about 100 μm.Activity of the enzyme is not affected by mevalonic acid at the concentrations tested (up to 1.0 mm). 3-Hydroxy-3-methylglutaric acid and free CoA cause substantial inhibition, whereas gibberellic acid has no effect.The activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase is twice as high in etiolated seedlings as in green seedlings. In green seedlings activity is highest in the apical bud, declines sharply in semimature leaves, and there is almost no activity in mature leaves.     

Release of glucose-6-phosphate dehydrogenase from cortex of Spisula eggs at fertilization and its recombination after meiosis

Changes in subcellular distributions of glucose-6-phosphate dehydrogenase (G6PDH) were observed after fertilization or artificial (KCl) activation of Spisula eggs. Though the total activity of G6PDH did not change during early stages, that in the 100,000g supernatant fraction increased after fertilization, attained a maximum at the first meiotic metaphase, and then decreased. This change of activity in the supernatant was accompanied by a mirror-image change of activity in the pellet. Most of the G6PDH was localized in the 3000g pellet fraction; furthermore, the activity in isolated cortices showed fluctuations during meiosis similar to that of the 3000g pellet fraction. Conditions for the release and binding of the NADP-specific G6PDH from the pellet fraction were investigated in vitro. NADP⁺ or NADPH can induce release of G6PDH, although NADPH is three to four times more efficient than NADP⁺. NAD⁺ does not affect release. High concentrations of salts (ionic strength >0.3) caused complete G6PDH release from the pellet. Although raising the pH alone showed only a slight releasing effect, increase of pH to pH 7 or above considerably augmented release due to NADP⁺ or NADPH. The release of G6PDH from the pellet fraction was shown to be reversible. These results suggest that the reversible association of G6PDH with particulate components of the cytoplasm may play an important role in regulation of G6PDH activity in marine eggs and that the cortex is one of the sites which may be involved in such regulation. The mechanism of recombination of G6PDH with its sites remains to be elucidated.     

Relative Activities and Characteristics of Some Oxidative Respiratory Enzymes from Conidia of Verticillium albo-atrum

Conidia of Verticillium albo-atrum Reinke and Berthold, collected from shake cultures grown in Czapek broth, were sonified for 4 or 8 minutes or ground frozen in a mortar to obtain cell-free homogenates. These were assayed for certain enzymes associated with respiratory pathways. Malic dehydrogenase was the most active, glucose-6-P and NADH dehydrogenase were less active, NADH-cytochrome c reductase, NADPH dehydrogenase, and cytochrome oxidase were low in activity, and succinic dehydrogenase and succinic cytochrome c reductase were very low to negligible in activity. No NADH oxidase activity was detected.

With the exception of NADH-cytochrome c reductase and possibly succinic dehydrogenase and cytochrome c reductase, there was no evident increase in specific activity of the enzymes during germination. Some NADH-cytochrome c reductase and a small amount of succinic-dehydrogenase and cytochrome c reductase were associated with the particulate fraction from 105,000 × g centrifugation. The other enzymes, including cytochrome oxidase, almost completely remained in the supernatant fraction.

Menadione and vitamin K-S(II) markedly stimulated NADH-cytochrome c reductase activity in the supernatant fraction but had much less effect on NADPH-cytochrome c reductase in this fraction or on either of these enzyme systems in the particulate fraction. Electron transport inhibitors affected particulate NADH- and NADPH-cytochrome c reductase activity but had no effect on these in the supernatant fraction.

     

3-Hydroxy-3-methylglutaryl CoA reductase of Hevea latex: The occurrence of a heat-stable activator in the C-serum

The effect of the C-serum (the cytosol) on the activity of 3-hydroxy-3-methylglutaryl CoA reductase in the latex of $\text{Hevea}\text{brasiliensis}$ was investigated. Depending on the clone from which the latex was obtained, the C-serum was found to depress or activate or have little effect on the enzyme activity. Boiling the C-serum however, resulted in a consistent activation effect in all the clones examined. Optimal activation was obtained with 20 μl boiled C-serum. Dialysis or EDTA (40 mM) treatment of the boiled C-serum did not diminish the activation effect. Although not essential, dithiothreitol complemented the activation effect of the boiled C-serum and the optimal concentration was 10 mM. Trypsin digestion of the boiled C-serum resulted in the complete loss of the activation effect. The activator in the boiled C-serum was salted out by ammonium sulphate at 25 – 100% saturation. Hevein had no effect on reductase activity.     

Aldrin epoxidation by plant root extracts

Aldrin epoxidase activity of the cell-free pea and bean root preparations was located in the particulate fraction. High speed centrifugation at 250 000 g for 2 hr resulted in a pellet with almost all the activity of the crude cell-free preparations. While the epoxidase was stimulated by NADPH generating system in these cell-free root preparations, that in high speed centrifugation pellets was not. Aldrin epoxidase activity of the dwarf bean root homogenates was increased by addition of p-aminobenzoic acid (10^?4 M). This increase in activity is above that already manifested by Polyclar AT. No activity was detected in the dormant or germinating Alaska peas or dwarf beans until the 5th and 6th day, respectively.     

Purification and Properties of a Diacetyl Reductase from Escherichia coli

A reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with the ability to reduce diacetyl has been isolated from Escherichia coli and has been purified 800-fold to near homogeneity. The product of the reduction of diacetyl was shown to be acetoin. The enzyme proved to catalyze the oxidation of NADPH in the presence of both uncharged α- and β-dicarbonyl compounds. Even monocarbonyl compounds showed slight activity with the enzyme. On the basis of its substrate specificity, it is suggested that the enzyme functions as a diacetyl reductase. In contrast to other diacetyl reductases, the one reported here is specific for NADPH and does not possess acetoin reductase activity. The pH optimum of this enzyme was found to be between 6 and 7. The maximal velocity for the NADPH-dependent reduction of diacetyl was determined to be 9.5 μmol per min per mg of protein and the K_m values for diacetyl and NADPH were found to be 4.44 mM and 0.02 mM, respectively. The molecular weight was estimated by gel filtration on Sephadex G-100 to be approximately 10,000.     

Some properties of purified 3-hydroxy-3-methylglutaryl coenzyme A reductase phosphatases from rat liver

Incubation of four purified rat liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase phosphatases (G. Gil, M. Sitges, and F. G. Hegardt, (1981) Biochim. Biophys. Acta663, 211–221) with HMG-CoA, CoA, NADPH, or citrate caused a concentration-dependent inactivation of the enzyme activities. HMG-CoA and CoA showed similar patterns of inactivation and at 0.5 mm of both compounds, the four reductase phosphatases were fully inhibited. Half-maximal inactivation was comprised between 0.02 and 0.1 mm of HMG-CoA and CoA. NADPH at concentration ranging between 5 and 10 mm produced complete inactivation of reductase phosphatases. Citrate at 5 mm produced full inactivation, and half-maximal inhibition ranged from 0.1 to 0.4 mm for the different phosphatases. The behavior of fluoride varied with respect to the four phosphatases: Low molecular forms were inactivated in a similar manner as described for other protein phosphatases. However, high molecular forms were slightly inactivated, and phosphatase IIa at 100 mm showed a level of activity similar to the control. The effect of KCl on the four reductase phosphatases could explain this behavior since at high concentrations, KCl (and NaCl) produced activation in both high and low molecular forms, this effect being more enhanced in high M_r reductase phosphatases. The insensitivity to fluoride of high M_r reductase phosphatases could explain the discrepancies in percentage of the active form of HMG-CoA reductase described previously in literature.     

Purification and characterization of methylglyoxal reductase from Hansenula mrakii

Methylglyoxal reductase was purified from Hansenula mrakii IFO 0895 to a homogenous state on polyacrylamide gel electrophoresis. The enzyme consisted of a single polypeptide chain with a molecular weight of 34,000. The enzyme was specific to methylglyoxal (K_m = 1.92 mM) and NADPH (K_m = 40.8 μM). The activity of the enzyme was inhibited by p-chloromercuribenzoate and HgCl₂. NADP also inhibited the activity of the enzyme, and the K_i value was calculated to be 0.25 mM.     

Biosynthesis of fatty alcohols,alkane-1,2-diols and wax esters in particulate preparations from the uropygial glands of white-crowned sparrows (Zonotrichia leucophrys)

Biosynthesis of sebaceous gland waxes was studied with the uropygial gland of the white-crowned sparrow as the experimental tissue. A 27,000g particulate preparation from this gland catalyzed reduction of palmitoyl-CoA to hexadecanol at an optimum pH near 5.0 with NADPH as the preferred reductant. At low protein concentrations, palmitoyl-CoA inhibited the reductase and bovine serum albumin prevented this inhibition. An apparent K_m of 0.3 mm was calculated for palmitoyl-CoA from linear double-reciprocal plots ignoring the inhibitory concentration of the substrate. An apparent K_m of 3 mm was calculated for NADPH from linear double-reciprocal plots. Palmitoyl-CoA reduction was inhibited by thiol directed reagents such as p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide. The particulate fraction also catalyzed esterification of hexadecanol with endogenous C₁₆ and C₁₈ acyl moieties with an optimum pH of 7.5. Stimulation of esterification of hexadecanol by ATP and CoA as well as by low concentrations of palmitoyl-CoA suggests that the CoA esters of fatty acids are involved in esterification. Tween-20 stimulated esterification of hexadecanol and hexadecyl dodecanoate was the major wax ester formed in the presence of Tween-20 suggesting that the C₁₂ acid of Tween-20 participated in esterification. Ignoring the inhibitory concentrations of hexadecanol (>0.2 mm), an apparent K_m of 0.1 mm was calculated from linear double-reciprocal plots. α-Hydroxylation of palmitic acid was demonstrated in cell-free extracts of the uropygial gland. A 27,000g particulate preparation from the gland catalyzed the reduction of α-hydroxypalmitic acid to hexadecane-1,2-diol with NADPH as the preferred reductant at an optimum pH near 6.5. This reduction required both ATP and CoA, suggesting that α-hydroxyacyl-CoA was the true substrate for the reductase. With stereospecifically labeled NADP³H, it was shown that both acyl-CoA reduction and α-hydroxy acid reduction involved transfer of the hydride specifically from the B-side of the nicotinamide ring of NADPH. Subcellular fractionation using sucrose density gradient centrifugation strongly suggested that the enzymes which catalyzed reduction of palmitoyl-CoA and α-hydroxypalmitic acid as well as the esterification of hexadecanol are localized in the microsomal membranes of the gland.     

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

Purification and characterization of sepiapterin reductase from rat erythrocytes

Sepiapterin reductase from rat erythrocyte hemolysate was purified 2000-fold to apparent homogeneity with 30% yield. The specific activity of the purified enzyme was 18 units/mg protein, and its molecular weight was 55 000. The enzyme consists of two identical subunits, each of which has a molecular weight of 27 500. The enzyme showed a single peak by isoelectric focusing with a pI of 4.9 and partial specific volume of 0.73 cm³/g. The amino acid composition was determined. pH optimum of the enzyme was 5.5. The equilibrium constant of 2.2·10⁹ of the enzyme showed that the equilibrium lies much in favor of dihydrobiopterin formation from sepiapterin in rat erythrocytes. From steady-state kinetic measurements, ordered bi-bi mechanism was proposed to the reaction of sepiapterin reductase in which NADPH binds to free enzyme and sepiapterin binds next. NADP⁺ is released after the release of dihydrobiopterin. The K_m values for sepiapterin and NADPH were 15.4 μM and 1.7 μM, respectively, and the V_max value was 21.7 μmol/min per mg.     

Glutathione s-transferase from hevea brasiliensis

Glutathione (GSH) S-transferase can be detected in a variety of tissues of Hevea brasiliensis. Lyophilized powders prepared from 20 000 g supernatants of latex adjusted to pH 5.0 contain substantial amounts of GSH S-transferase activity which is stable at ? 20° for up to 6 months. The enzyme has a broad pH optimum between 8.5 and 9.5. The K_m values for GSH and 1-chloro-2,4-dinitrobenzene are in the range of 33–45 and 150–200,μM, respectively. The enzyme has a MW in the range of 47 000–50 000 and an isoelectic point of 4.3. Although it appears homogeneous on analytical polyacylamide disc gel electrophoresis (PAGE) and isoelectric focusing, it resolves into five forms on DEAE-Sephadex chromatography.     

Affinity purification and properties of porcine brain aldose reductase.

Aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) has been purified 1500-fold from porcine brain in a four-step procedure employing Blue-Sepharose 6B affinity chromatography. The purified enzyme was shown to be apparently homogeneous by polyacrylamide gel electrophoresis. The enzyme is a single chain polypeptide of molecular weight 40 000, pH optimum 5.0 K(app)(xylose) 4 mM; K(app)(NADPH) 3 microM. The relative substrate activities, activation with sulfate ion, and limited oxidative and NADH-related reductive activities confirm the classification of this enzyme as aldolase reductase. The activity of the reductase with p-nitrobenzaldehyde and 3-indolacetaldehyde and the similarity of its physical properties with the 'low Km' aldehyde reductase of porcine brain previously reported indicates that these enzymes may be identical.     

Biosynthetic and metabolic activities of some organelles in Papaver somniferum latex

The biosynthesis of the five major alkaloids of the opium poppy (Papaver somniferum L. from radioactive dihydroxyphenylalanine has been studied in the 1000 g, 10 000 g, 100 000 g pellets and the 100 000 g supernatant fractions of the capsule latex. A normal poppy variety as well as one which produces only traces of alkaloids were used. Definite evidence of biosynthesis was obtained for both varieties but only in the 1 000 g pellet (as previously reported . None was found in the other fractions although electron microscopy showed that organelles, including vesicles, were present. The amounts of alkaloid biosynthesized however were very small relative to the amounts involved in the rapid changes already reported for the developing capsules. In contrast, all fractions of the latex were able to metabolize T-morphine in vitro, with the 100 000 g supernatant showing the highest activity and the amounts involved were also consistent with the changes found in the living plant.