Product Feedback Inhibition Research Articles

Interaction of acyl-CoA binding protein (ACBP) on processes for which acyl-CoA is a substrate, product or inhibitor.

It is shown that acyl-CoA binding protein (ACBP), in contrast with fatty acid binding protein (FABP), stimulates the synthesis of long-chain acyl-CoA esters by mitochondria. ACBP effectively opposes the product feedback inhibition of the long-chain acyl-CoA synthetase by sequestration of the synthesized acyl-CoA esters. Feedback inhibition of microsomal long-chain acyl-CoA synthesis could not be observed, due to the formation of small acyl-CoA binding vesicles during preparation and/or incubation. Microsomal membrane preparations are therefore unsuitable for studying feedback inhibition of long-chain acyl-CoA synthesis. ACBP was found to have a strong attenuating effect on the long-chain acyl-CoA inhibition of both acetyl-CoA carboxylase and mitochondrial adenine nucleotide translocase. Both processes were unaffected by the presence of long-chain acyl-CoA esters when the ratio of long-chain acyl-CoA to ACBP was below 1, independent of the acyl-CoA concentration used. It is therefore not the acyl-CoA concentration as such which is important from a regulatory point of view, but the ratio of acyl-CoA to ACBP. The cytosolic ratio of long-chain acyl-CoA to ACBP was shown to be well below 1 in the liver of fed rats. ACBP could compete with the triacylglycerol-synthesizing pathway, but not with the phospholipid-synthesizing enzymes, for acyl-CoA esters. Furthermore, in contrast with FABP, ACBP was able to protect long-chain acyl-CoA esters against hydrolysis by microsomal acyl-CoA hydrolases. The results suggest that long-chain acyl-CoA esters synthesized for either triacylglycerol synthesis or beta-oxidation have to pass through the acyl-CoA/ACBP pool before utilization. This means that acyl-CoA synthesized by microsomal or mitochondrial synthetases is uniformly available in the cell. It is suggested that ACBP has a duel function in (1) creating a cytosolic pool of acyl-CoA protected against acyl-CoA hydrolases, and (2) protecting vital cellular processes from being affected by long-chain acyl-CoA esters.

GTP‐specific pyrophosphorylation of thiamin in dark‐grown soybean (Glycine max) seedling axes

ATP:thiamin pyrophosphotransferase (TPT: EC 2.7.6.2) was purified 5 900‐fold from 48 h dark‐grown soybean [Glycine max (L.), Merr. cv. Ransom II] seedling axes. TPT activity was monitored during purification by measuring the formation of thiamin pyrophosphate (TPP) from [2‐14C]‐thiamin at optimal pH (7.3). Although other nucleoside triophosphates were active as pyrophosphate donors (apparent Kms from 21 to 138 mM), GTP was the preferred nucleotide with an apparent Km of 0.021 mM. TPT activity was extremely sensitive to TPP formation, suggesting product feedback inhibition of TPT activity in vivo. Sulfhydryl, H+ and Mg2+ concentrations, either independently or in concert, were found to affect TPT activity.

Biosynthesis of the Tetrapyrrole Pigment Precursor, δ-Aminolevulinic Acid, from Glutamate

delta-Aminolevulinic acid (ALA), the common biosynthetic precursor of hemes, chlorophylls, and bilins, is synthesized by two distinct routes. Among phototrophic species, purple nonsulfur bacteria form ALA by condensation of glycine with succinyl-CoA, catalyzed by ALA synthase, in a reaction identical to that occurring in the mitochondria of animals, yeast, and fungi. Most or all other phototrophic species form ALA exclusively from the intact carbon skeleton of glutamic acid in a reaction sequence that begins with activation of the alpha-carboxyl group of glutamate by an ATP-dependent ligation to tRNA(Glu), catalyzed by glutamyl-tRNA synthetase. Glutamyl-tRNA is the substrate for a pyridine nucleotide-dependent dehydrogenase reaction whose product is glutamate-1-semialdehyde or a similar reduced compound. Glutamate-1-semialdehyde is then transaminated to form ALA. Regulation of ALA formation from glutamate is exerted at the dehydrogenase step through end product feedback inhibition and induction/repression. In some species, end product inhibition of the glutamyl-tRNA synthetase step and developmental regulation of tRNA(Glu) level may also occur.

Glucocorticoids both stimulate and inhibit production of pulmonary surfactant protein A in fetal human lung.

Pulmonary surfactant is a mixture of phospholipids and proteins which stabilizes lung alveoli and prevents respiratory failure. The surfactant-associated protein of Mr = 28,000-36,000 (SP-A) influences the structure, function (film formation), and metabolism of surfactant. We have characterized glucocorticoid regulation of SP-A and SP-A mRNA in explants of fetal human lung. The time course of response to dexamethasone was biphasic, with early stimulation and later inhibition of SP-A accumulation. Maximal induction of SP-A occurred with 3-10 nM dexamethasone and approximately 300 nM cortisol for 72 hr, and stimulation diminished at higher concentrations. SP-A mRNA accumulation was maximally stimulated at 24-48 hr of exposure to dexamethasone (10 nM) and was generally inhibited by 4-6 days. Stimulation was also observed with cortisone and corticosterone but not with sex steroids, suggesting a receptor-mediated process. When explants were exposed to cortisol for only 24 hr, SP-A content was transiently increased above the level in continuously treated tissue and subsequently was similar to control. The content of SP-A and its mRNA was also increased by dibromo-cAMP, terbutaline, and forskolin, and effects were approximately additive with those of dexamethasone. However, elevated in tracellular cAMP did not alter the biphasic time course or dose-response patterns of dexamethasone. We propose that glucocorticoids have both stimulatory and inhibitory effects on SP-A gene expression. This biphasic regulation is not consistent with generalized toxic effects, product-feedback inhibition, or receptor down-regulation, and it appears to be specific for SP-A among the various surfactant components.

Regulation of indices of cholesterol synthesis in human mononuclear leukocytes by dietary cholesterol and fat saturation

The responses of 2 indices of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase activity and incorporation of [ 14C]acetate into sterols, in mononuclear leukocytes freshly isolated from peripheral blood to variation in the ratio of saturated to unsaturated fat (S: U) and the amount of cholesterol absorbed from the diet were examined in 24 free-living men. Increasing S : U was associated with increasing plasma cholesterol level ( r = 0.27, P = 0.03) and increasing reductase activity in leukocytes ( r = 0.60, p < 0.00'1). This finding is consistent with the hypothesis that saturated fat decreases the flux of cholesterol from plasma into cells thereby releasing reductase from product feedback inhibition. Reductase activity, after controling for the effect of S : U, was negatively correlated with absorbed cholesterol from sources other than eggs ( r = − 0.42, P = 0.02). Surprisingly, change in reductase activity was positively correlated with change in absorbed cholesterol upon eating eggs ( r = 0.49, P = 0.008). Sterol labeling was negatively correlated with absorbed cholesterol from all sources including eggs ( r = −0.64, P < 0.001) and was uncorrelated with S : U. Reductase activity and sterol labeling responded in parallel to cholesterol in foods other than eggs but not to egg feeding nor to S : U, thus it is unclear which test best reflects engogenous sterol synthesis in these cells.

Influence of 20 alpha-dihydroprogesterone on progesterone output by human chorion explants.

The output of progesterone by explants of chorion incubated without exogenous precursor was increased in the presence of 20 alpha-dihydroprogesterone (20 alpha-diHP). We have now examined the nature of the 20 alpha-diHP effect. Explants of chorion were incubated with [3H]progesterone and 3 microM non-radiolabelled 20 alpha-diHP to examine the effect of 20 alpha-diHP on progesterone metabolism. Significantly more radioactivity remained as unmetabolized [3H]progesterone compared to chorion incubated in the absence of non-radiolabelled 20 alpha-diHP. There was also a decrease in the output of [3H]-20 alpha-diHP by the explants but no change in the output of a second metabolite of progesterone [3H]-5 alpha-pregnanedione. When explants were incubated with 20 alpha-diHP in the presence of the 3 beta-hydroxysteroid dehydrogenase inhibitor, trilostane, the output of progesterone was significantly greater than with explants incubated with trilostane but without addition of 20 alpha-diHP. Radiochemically pure [3H]progesterone was produced by explants of chorion incubated with [3H]-20 alpha-diHP. We conclude that 20 alpha-diHP may serve as substrate for chorionic progesterone production through 20 alpha-hydroxysteroid dehydrogenase. 20 alpha-diHP, formed from progesterone, may also exert product feedback inhibition on this enzyme.

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a K m for methionine of about 3–12 μM. In selected lines we have demonstrated a requirement for Mg 2+ in addition to that needed to form the Mg·ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.

Evidence for two distinct forms of fatty acid cyclooxygenase in brain.

The enzymatic metabolism of [14C]arachidonic acid (AA) was studied with microsomes prepared from rabbit medulla. Prostaglandin E2 (PGE2) levels, measured either by radiochemistry or radioimmunoassay, rose rapidly and abruptly plateaued within 5 min, while prostaglandin F2 alpha (PGF2 alpha) levels continued to rise for 30 min. The rapid termination of PGE2 biosynthesis was not the result of limited cofactor, substrate, or product feedback inhibition, nor was it due to PGE2-9-ketoreductase activity. Inhibition of the PGH2 leads to PGE2 isomerase by arachidonic acid or its metabolites could not explain the abrupt half in PGE2 biosynthesis. Proof for two separate cyclooxygenases comes from our observation that a preincubation of the brain microsomes with unlabeled AA eliminated PGE2 biosynthesis while PGF2 alpha production continued. Further evidence to suggest two cyclooxygenases in brain is derived from the observation that indomethacin inhibited PGE2 production at concentrations that did not affect PGF2 alpha biosynthesis. These results suggest that one fatty acid cyclooxygenase is closely associated with PGH2 leads to PGE2 isomerase and readily undergoes autodestruction and the second cyclooxygenase is associated with a PGH2 leads to PGF2 alpha reductase and is somewhat resistant to arachidonate-induced destruction and to nonsteroidal antiinflammatory agents.

Tyrosine hydroxylase. Activation by protein phosphorylation and end product inhibition.

Protein phosphorylation activates tyrosine hydroxylase in crude extracts of rat striatum, hypothalamus, and adrenal glands by a reduction in the apparent Km value for 6-methyltetrahydropterin. Removal of endogenous catecholamines by gel filtration or cation exchange results in a similar activation. Phosphorylation causes only a small additional reduction in the apparent Km for reduced pterin in striatal extracts from which catecholamines have been removed. Kinetic analysis indicates that protein phosphorylation causes a significant increase in the Ki for end product dopamine, whereas gel filtration or cation exchange treatment has little effect on the dopamine Ki value. None of the above treatments appears to change the molecular weight of the enzyme. At physiological concentrations of dopamine, the increase in Ki by phosphorylation would effectively release tyrosine hydroxylase from end product feedback inhibition.

Leishmania tarentolae: Proline anabolism in promastigotes

Using 14C-labeled substrates in conjunction with thin-layer Chromatographic radioautography, studies were done to determine the precursors for proline synthesis utilized by promastigotes of Leishmania tarentolae. The only substances in Trager's Defined Medium capable of acting as proline precursors under the conditions studied were l-arginine and l-ornithine. None of the other substrates tested (16 amino acids, five purines and pyrimidines, and glucose) were converted to proline. l-glutamate, l-aspartate, and d-glucose did not act as precursors for the imino acid although they stimulated oxygen uptake better than did l-arginine and l-ornithine. Proline synthesis was unaffected by the presence of preformed proline indicating the absence of end product feedback inhibition.

In vivo regulation of intermediate reactions in the pathway of tryptophan biosynthesis in Neurospora crassa.

The in vivo regulation of intermediate reactions in the pathway of tryptophan synthesis in Neurospora crassa was examined in a double mutant (tr-2, tr-3) which lacks the functions of the first and last enzymes in the pathway from chorismic acid to tryptophan. The double mutant can convert anthranilic acid to indole and indole-3-glycerol, and the production of these indolyl compounds by germinated conidia was used to estimate the activity of the intermediate enzymes in the pathway. Indole-synthesizing activity was maximal in germinated conidia obtained from cultures in which the levels of l-tryptophan were growth-limiting; the formation of this activity was markedly repressed when the levels of l-tryptophan exceeded those required for maximal growth. d-, 5-methyl-dl-, and 6-methyl-dl-tryptophan were less effective than l-tryptophan, and 4-methyl-dl-tryptophan, tryptamine, and indole-3-acetic acid were ineffective in repressing the formation of indole-synthesizing activity; anthranilic acid stimulated the formation of indole-synthesizing activity. Preformed indole-synthesizing activity was strongly and specifically inhibited by low levels of l-tryptophan; several related compounds were ineffective as inhibitors. These results suggest that, in addition to repression, an end product feedback inhibition mechanism is operative on an intermediate enzyme(s) in tryptophan biosynthesis. The relation of these results to other in vivo and in vitro studies and to general aspects of the regulation of tryptophan biosynthesis in N. crassa are discussed.

Acyl Derivatives of Homoserine as Substrates for Homocysteine Synthesis in Neurospora crassa, Yeast, and Escherichia coli

Abstract Activated forms of l-homoserine have been shown to be more readily utilized as substrates than homoserine for the homocysteine synthetases (enzymes which catalyze the formation of homocysteine from homoserine and sulfide in the presence of pyridoxal phosphate) of Neurospora crassa, yeast, and Escherichia coli KB. This substrate preference is shown by the increased specific activities of the enzyme reactions. Specificity for the type of acylated derivative of homoserine is apparent. Enzymes from the fungi preferentially utilize O-acetyl-l-homoserine, and the enzyme from the bacterium requires O-succinyl-l-homoserine. Methionineless mutants of Neurospora contain homocysteine synthetases with specific activities nearly equivalent to those of the wild type when the substrate is O-acetylhomoserine. End product feedback inhibition by l-methionine on the homocysteine synthetases, when the substrates used were acyl derivatives of homoserine, is described for the enzymes from Neurospora and yeast, but was not demonstrated with the enzyme from E. coli. O-Acetyl-l-serine is a required substrate for cysteine synthesis by an enzyme from E. coli KB. Some preference for O-acetylserine was exhibited by the enzyme from yeast; however, cysteine synthetase from Neurospora showed no marked preference for this compound over serine.