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J W Porter

Publications and source records attributed to J W Porter.

At least 73 records · Page 4Linked to original sources
PubMed1977

Dissociation of prelycopersene pyrophosphate synthetase from phytoene synthetase complex of tomato fruit plastids.

The partially purified phytoene synthetase enzyme complex obtained from tomato fruit plastids dissociates into two or more subunit species on chromatography in low ionic strength buffer on DEAE-cellulose. One of these subunits prelycopersene pyrophosphate synthetase, has a molecular weight of approximately 40,000, whereas the phytoene synthetase complex has a molecular weight of 200,000. The prelycopersene pyrophosphate synthetase catalyzes the conversion of isopentenyl pyrophosphate to geranylgeranyl and prelycopersene pyrophosphates. The identities of these substances were established by thin layer chromatography in several solvent systems. The formation of both geranylgeranyl and prelycopersene pyrophosphates by this enzyme supports earlier results with cruder enzyme systems which suggested that these compounds are intermediates in the synthesis of phytoene.

Carotenoids↗
PubMed1977

Purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver.

A procedure for the purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating); EC 1.1.1.34] solubilized from rat liver microsomes is reported. This enzyme has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This represents a 4100-fold purification over the activity in microsomes, and a specific activity that is approximately 20-fold greater than the highest previously reported value. The enzyme is judged to be homogeneous on the basis of sodium dodecyl sulfate/polyacrylamide disc gel electrophoresis, polyacrylamide disc gel electrophoresis, and immunoanalysis. Data are also presented that indicate the separation of enzymatically active and inactive species of 3-hydroxy-3-methylglutaryl-coenzyme A reductase on affinity chromatography on a coenzyme A column.

Alcohol Oxidoreductases↗
PubMed1976

Purification of beta-hydroxy-beta-methylglutaryl-coenzyme A reductase from yeast.

Beta-Hydroxy-beta-methylglutaryl-coenzyme A reductase of yeast has been solubilized by two different methods and then purified approximately 5000-fold. The purified enzyme shows a single precipitin band on immunodiffusion, and it moves as a single band of protein and enzyme activity on gel filtration and diethylaminoethylcellulose column chromatography. It also shows one major band on polyacrylamide gel electrophoresis. The specific activity of the pure enzyme is 18 000 to 22 000 nmol of reduced nicotinamide adenine dinucleotide phosphate oxidized per min per mg of protein. The molecular weights of the enzyme, estimated by gel filtration, and the subunits, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, are 2.6 X 10(5) and 6.0 X 10(4), respectively.

Alcohol Oxidoreductases↗
PubMed1976

Kinetic analysis of the individual reductive steps catalyzed by beta-hydroxy-beta-methylglutaryl-coenzyme A reductase obtained from yeast.

The mechanism of action of yeast beta-hydroxy-beta-methylglutaryl-coenzyme A reductase has been investigated through kinetic studies on the oxidation of mevaldate by nicotinamide adeninine dinucleotide phosphate (NADP) in the presence of coenzyme A (CoA) and on the reduction of mevaldate by reduced NADP (NADPH) in the absence of presence of CoA or acetyl-CoA. NADP and mevalonate were also used as product inhibitors of the reduction of mevaldate. In the reduction of mevaldate to mevalonate, coenzyme A and acetyl-CoA decreased the Km for mevaldate 30- and 3-fold, respectively. Both compounds increased the Vmax 1.5-fold. These results suggest that CoA is an allosteric activator for the second reductive step and that it acts by enhancing the binding of mevaldate. The intersecting patterns obtained from initial velocities and the patterns produced by product inhibitions suggest the following features of the mechanism. The binding of substrates and release of products proceeds sequentially in both reductive steps, and is ordered throughout or random with respect to the binding of the beta-hydroxy-beta-methylglutaryl-coenzymeA and the first NADPH. The binding of NADPH enhances the binding of the beta-hydroxy-beta-methylglutaryl portion of the CoA ester and the binding of free mevaldate, whereas the binding of NADP leads to an increased affinity of the enzyme for the hemithioacetal (of mevaldate and CoA) and for mevalonate. Thus, the replacement of NADP by NADPH after the first reductive step promotes the conversion of the hemithioacetal to the free carbonyl form, which is then rapidly reduced. The products, CoA and mevalonic acid, of the second reductive step leave the enzyme before the release of the second NADP. This release of the last product is probably the rate-limiting step for the overall process.

Alcohol Oxidoreductases↗
PubMed1976

Enzymatic synthesis of C40 carotenes by cell-free preparation from Halobacterium cutirubrum.

[14C]Mevalonate or (14C)isopentenyl pyrophosphate was found to be converted to transphytoene, trans-phytofluene, lycopene, and beta-carotene by a cell-free 270 000 X g supernatant fraction prepared from Halobacterium cutirubrum cells that were broken by manual grinding with glass beads. Incubations were done under N2 in the dark at 37 degrees C in 4 M NaCl in presence of FAD, NADP, and MgCl2; ATP was also added when mevalonate was the substrate. This system was also capable of converting trans-(14C)phytoene to beta-carotene via the intermediates trans-phytofluene, zeta-carotene, neurosporene, lycopene, and gamma-carotene. Each of these labelled intermediates on incubation separately with the same enzyme system was shown to be converted to the intermediates farther down the pathway. The results of this study show that the biosynthetic pathway for the formation of C40 carotenes in H. cutirubrum proceeds as follows: isopentenyl pyrophosphate leads to trans-phytoene leads to trans-phytofluene leads to zeta-carotene leads to neurosporene leads to lycopene leads to gamma-carotene leads to beta-carotene. This pathway differs from that in higher plants in that the cis isomers of phytoene and phytofluene are not on the main pathway of carotene biosynthesis, as they are in higher plants. Furthermore, trans-phytoene, which has not been reported to have any role in higher plants, appears to be the main intermediate in carotene biosynthesis in H. cutirubrum.

Carotenoids↗
PubMed1975

Subunits of fatty acid synthetase complexes. Enzymatic activities and properties of the half-molecular weight nonidentical subunits of pigeon liver fatty acid synthetase.

The separation of the half-molecular weight, nonidentical subunits (I and II) of the pigeon liver fatty acid synthetase complex has been achieved on a large (20 mg) scale by affinity chromatography on Sepharose epsilon-aminocaproyl pantetheine. This separation requires a careful control of temperature, ionic strength, pH, and column flow rate for success. The yield of subunit II is further improved by transacetylation (with acetyl-CoA) of the dissociated fatty acid synthetase prior to affinity chromatography. The separated subunit I (reductase) contains the 4'-phosphopantetheine (A2) acyl binding site, two NADPH binding sites, and beta-ketoacyl and crotonyl thioester reductases. Subunit II (transacylase) contains the B1 (hydroxyl or loading) and B2 (cysteine) acyl binding sites, and acetyl- and malonyl-CoA: pantetheine transacylases. When subunit I is mixed in equimolar quantities with subunit II, an additional NADPH binding site is found even though subunit II alone shows no NADPH binding. Both subunits contain activities for the partial reactions, beta-hydroxybutyryl thioester dehydrase (crotonase) and palmityl-CoA deacylase. Subunit I has 8 sulfhydryl groups per mol whereas subunit II has 60. Reconstitution of fatty acid synthetase activity to 75% of the control level is achieved on reassociation of subunits I and II.

Acetyltransferases↗
PubMed1975

Fatty acid synthetase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from pigeon liver.

The kinetic mechanism of pigeon liver fatty acid synthetase action has been studied using steady state kinetic analysis. Initial velocity studies are consistent with an earlier suggestion that the enzyme catalyzes this reaction by a seven-site ping-pong mechanism. Although the range of substrate concentrations that could be used was limited by several factors, the initial velocity patterns showing the relationship between the substrates acetyl coenzyme CoA, malonyl-CoA, and NADPH appear to be a series of parallel lines, regardless of which substrate is varied at fixed levels of a second substrate. However, two of the substrates, acetyl-CoA and malonly-CoA, apparently exhibit a competitive substrate inhibition with respect to each other, but NADPH shows no inhibition of any kind. Product inhibition patterns suggest that free CoA is competitive versus acetyl-CoA and malonyl-CoA and is uncompetitive versus NADPH, and that NADP+ is competitive versus NADPH and uncompetitive versus acetyl-CoA or malonyl-CoA. These results are consistent with a seven-site ping-pong mechanism with intermediates covalently bound to 4'-phosphopantetheine (part of acyl carrier protein). Double competitive substrate inhibition by acetyl-CoA and malonyl-CoA is consistent with the rate equation derived for the over-all mechanism. The kinetic mechanism developed from these results is capable of explaining the formation of fatty acids from malonyl-CoA and NADPH alone (Katiyar, S. S., Briedis, A. V., and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420) and also the formation of triacetic acid lactone from either malonyl-CoA alone or acetyl-CoA plus malonyl-CoA.

Acetyl Coenzyme A↗
PubMed1975

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.

Animals↗