共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
3.
C M Nepokroeff A A Qureshi J W Porter 《Biochemical and biophysical research communications》1975,67(1):345-352
Pigeon liver fatty acid synthetase proteins (apo- and holo-forms) have been synthesized in a cell-free system reconstituted from polysomes and a soluble enzyme fraction. Identification of the cell-free synthesized products as fatty acid synthetase was achieved by affinity chromatography, by immuno-precipitation and by the simultaneous conversion of both the authentic carrier protein and the synthesized products from the holo- to the apo-form of the synthetase. The reverse conversion was also effected. 相似文献
4.
The synthesis of the multienzyme complex rat liver fatty acid synthetase was investigated utilizing modifications of methods developed in the laboratory of Schimke (Schimke, R. T. (1964) J. Biol. Chem. 239, 3808-3817 and Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). The relative amounts of radioactivity from a pulse of labeled lysine appearing in polypeptides derived from purified synthetase complex can be measured compensating for the varying amounts of lysine per polypeptide chain. The results show that labeled amino acid is incorporated into polypeptides derived from the complex at heterogeneous rates. However, 10 to 15 hours after the administration of a pulse, the amount of label per lysine residue in these polypeptides is identical. The results support the previously proposed model of this multienzyme complex (Tweto, J., Dehlinger, P., and Larrabee, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1377). The previous work and that reported here suggests the existence of a pool of synthetase subunits which is an obligatory intermediate in both synthesis and turnover of the complex. The results obtained in this work are consistent with this model if the exchange of subunits into the intact complex is a relatively slow process requiring several hours to reach equilibrium. 相似文献
5.
6.
7.
8.
Activation of liver pyruvate kinase by fructose 1,6-diphosphate. 总被引:2,自引:0,他引:2
9.
S Kumar 《The Journal of biological chemistry》1975,250(13):5150-5158
Fatty acid synthetase complex (Mr = 500,000) purified from pigeon liver homogenates is inactivated by phenylmethylsulfonyl fluoride. A well characterized inhibitor of serine esterases. Pseudounimolecular kinetics are followed at all inhibitor concentrations studied (0.05 to 1.0 mM). The second order rate constant obtained at pH 7.0, 30 degrees in 0.05 M potassium phosphate, 1 mM EDTA is 250 plus or minus 10 M-1 min-1 and appears to be independent of pH between 6 and 7.9. The inactivation of the enzyme complex appears to be selective since only one of the several component enzymes of fatty acid synthesis, palmityl-CoA deacylase, is inhibited. Acetyl- and malonyl-CoA-pantetheine transacylase activities as well as the kinetics of the reduction and dehydration steps are nearly identical for the native and the modified enzymes. The rate of approach of the condensation-CO2 exchange reaction (substrates: hexanoyl-CoA, malonyl-CoA, CoA, and H14CO3-) is slightly slower in the modified enzyme, though this change is not large enough to account for total loss of activity for fatty acid synthesis. The rate of loss of palmityl-CoA deacylase activity at a constant inhibitor concentration follows biphasic kinetics. Complete inactivation is achieved only after 2 mol of the inhibitor are bound per mol of the enzyme complex. Acetyl-, butyryl-, and hexanoyl-CoA thioesters (at 1.0 mM concentrations) protect the enzyme complex against inactivation by phenylmethylsulfonyl fluoride whereas CoA has no effect. Malonyl-CoA on the other hand, promotes inhibitor-mediated inactivation. Of the N-acetyl cysteamine derivatives tested, S-acetyl-N-acetyl cysteamine (at 10 mM) gives almost complete protection against inactivation whereas S-acetoacetyl-, S-beta-hydroxybutyryl-, and S-crotonyl-N-acetyl cysteamine thioesters exhibit either slight or no protection. These data demonstrate that phenylmethylsulfonyl fluoride is a selective reagent for the inactivation of functional fatty acyl deacylase component(s) of the pigeon liver fatty acid synthetase complex, and that it has no effect on malonyl or acetyl transacylases. The data are also in accord with the postulation that the inhibitor interacts at two catalytic centers of the enzyme complex. Furthermore, the patterns of protective effects shown by saturated acyl-CoA asters and malonyl-CoA point to different mechanisms of deacylation for these esters. 相似文献
10.
11.
12.
13.
Pigeon liver fatty acid synthetase (FAS) was inactivated irreversibly by stoichiometric concentration of o-phthalaldehyde exhibiting a bimolecular kinetic process. FAS-o-phthalaldehyde adduct gave a characteristic absorption maxima at 337 nm. Moreover this derivative showed fluorescence emission maxima at 412 nm when excited at 337 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The inactivation is caused by the reaction of the phosphopantetheine -SH group since it is protected by either acetyl- or malonyl-CoA. The enzyme incubated with iodoacetamide followed by o-phthalaldehyde showed no change in fluorescence intensity but decrease in intensity was found in the treatment of 2,4,6-trinitrobenzenesulphonic acid (TNBS), a lysine specific reagent with the enzyme prior to o-phthalaldehyde addition. As o-phthalaldehyde did not inhibit enoyl-CoA reductase activity, so nonessential lysine is involved in the o-phthalaldehyde reaction. Double inhibition experiments showed that 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a thiol specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 moles of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation. 相似文献
14.
15.
Synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase 总被引:1,自引:0,他引:1
Pigeon liver fatty acid synthetase has been found to catalyze the formation of palmitic acid from malonyl-CoA and NADPH in the absence of acetyl-CoA. Radio-chemical and spectral assays show that the activity of the complex in the absence of acetyl-CoA is about 25–30% of the activity in the presence of this compound. Initial velocities were determined for a series of reactions in which the malonyl-CoA concentration was varied over a range of 5–200 μm at a fixed NADPH concentration of 100μm and vice versa. No inhibitory effects of one substrate over the other were found. However, when the synthesis of fatty acids was studied in the presence of acetyl-CoA, a significant inhibitory effect of malonyl-CoA was observed. It has also been shown that the fatty acid synthetase synthesizes triacetic lactone from malonyl-CoA in the absence of NADPH and acetyl-CoA. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. Hence it is proposed that decarboxylation of the malonyl moiety bound covalently to 4′-phosphopantetheine occurs to yield acetyl-4′-phosphopantetheine. Further, it is proposed that the acetyl moiety of the latter compound is transferred to the cysteine site of the enzyme complex and that fatty acid synthesis proceeds in the presence of NADPH as proposed by Phillips et al. [Arch. Biochem. Biophys.138, 380 (1970)]. In the absence of NADPH triacetic lactone is formed. 相似文献
16.
Studies on the interaction between rabbit liver pyruvate kinase and its allosteric effector fructose 1,6-diphosphate 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Preparation of the L form of rabbit liver pyruvate kinase (EC 2.7.1.40) in the presence of fructose 1,6-diphosphate yielded an enzyme which was kinetically identical with the M or muscle-type form of pyruvate kinase found in liver. Chromatographic and dialysis studies of this complex showed that most of the fructose 1,6-diphosphate molecules were loosely bound to the enzyme, but dilution-dissociation studies and binding experiments established that there was a high initial affinity between the enzyme and fructose 1,6-diphosphate (K(assoc.)=2.3x10(9)), and that binding of the loosely bound fructose 1,6-diphosphate was concentration-dependent and a necessary condition to overcome the co-operative interaction observed with the homotropic effector phosphoenolpyruvate. Preparation of the liver enzyme in the absence of EDTA did not yield a predominantly M form of the enzyme, and incubation of the M form in the presence of EDTA did not convert it into the L form, but resulted in inhibition of enzyme activity. Immunological studies confirmed that the L and M forms in liver were distinct, and that preparation of the L form in the presence of fructose 1,6-diphosphate did not produce an enzyme antigenically different from the L form prepared in the absence of this heterotropic effector. 相似文献
17.
18.
O Brenner-Holzach 《Archives of biochemistry and biophysics》1979,194(2):321-327
The amino acid composition and other properties of fructose 1,6-diphosphate aldolase from pupae of Drosophila melanogaster are reported and compared with those of other class I aldolases. Drosophila aldolase subunits contain only four residues of cysteine, five histidines, and two methionines. All four cysteine side chains react with 5,5′-dithiobis(2-nitrobenzoic acid) only in the presence of denaturating agent and are therefore thought to be buried within the molecule. With bromoacetate one carboxymethyl group is incorporated in the native enzyme with the loss of 90% of catalytic activity; inorganic phosphate is partially inhibiting this reaction. The near-uv absorption spectra of Drosophila and rabbit muscle aldolases are similar, the insect enzyme having higher absorbancies over the entire region corresponding to its higher tryptophan content. Circular dichroism-spectra of Drosophila aldolase indicate an α-helix content of 26%. Both the insect and vertebrate enzymes display marked tryptophan ellipticity bands between 290 and 300 nm. 相似文献
19.
20.
Studies on the mechanism of the effects of experimental inflammation on adaptive synthesis of rat liver fatty acid synthetase 总被引:1,自引:0,他引:1
The mechanism of suppression, by experimental inflammation of the usual increase in hepatic fatty acid synthetase activity resulting from fat-free feeding following starvation (adaptive synthesis), was investigated immunochemically. That suppression results from changes in amount of hepatic fatty acid synthetase was shown by the observation that fatty acid synthetase preparations from inflamed and uninflamed animals, exhibiting a wide variety of specific enzyme activities, had identical immunochemical equivalence points. In confirmation of this, the amounts of fatty acid synthetase, determined by radial immunodiffusion in gels containing anti-fatty acid synthetase serum, varied concomitantly with changes in enzyme activity regardless of the relative times of inflammation and onset of adaptive synthesis. Serum insulin levels were not dramatically elevated during the first 48 h of fat-free feeding, but rose markedly thereafter. Inflammation, either alone or combined with fat-free feeding, resulted in increased serum glucose levels, followed by a similar pattern of increased serum insulin levels some 12 h later. Fat-free feeding did not affect serum cortisol levels, but depressed liver cyclic AMP. Inflammation invariably resulted in a marked increase in serum cortisol within 12 h and a concomitant elevation of hepatic cyclic AMP, indicating possible roles for cortisol and cyclic AMP in suppression of hepatic fatty acid synthetase synthesis. 相似文献