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Biomedical subjects

A D McCarthy

Publications and source records attributed to A D McCarthy.

27 records · Page 2Linked to original sources
PubMed1983

The multifunctional polypeptide chain of rabbit mammary fatty acid synthase contains a domain homologous with the acyl carrier protein of Escherichia coli.

The phosphopantetheine thiol of rabbit mammary fatty acid synthase was specifically alkylated using chloro[14C]acetyl-CoA and a radioactive fragment generated by limited elastase digestion of the modified protein was purified by gel filtration. We have previously mapped this fragment to an internal location in the 250 000-Mr polypeptide adjacent to the thioesterase domain [Eur. J. Biochem. 130, 185-193 (1983)]. The purified fragment had apparent molecular weights of 23 000 by gel filtration and 10 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while amino acid analysis indicated a minimal molecular weight of 10 400. We have determined the amino acid sequence of the first 64 residues of the fragment. The phosphopantetheine moiety is esterified to a serine at residue 38 in the sequence. When the sequences of the rabbit acyl carrier fragment and the 8847-Mr acyl carrier protein of Escherichia coli are aligned, 17 out of 64 residues are identical. These results suggest that the limited proteolysis delineates an internal acyl carrier domain within the rabbit protein and provide the first clear evidence that multifunctional fatty acid synthases have arisen by fusion of ancestral monofunctional proteins.

Acyl Carrier Protein↗
PubMed1983

Evidence that the multifunctional polypeptides of vertebrate and fungal fatty acid synthases have arisen by independent gene fusion events.

The enoyl reductase (NADPH binding site) of rabbit mammary fatty acid synthase has been radioactively labelled using pyridoxal phosphate and sodium [3H]borohydride. Using this method we have been able to add this site to the four sites whose location has already been mapped within the multifunctional polypeptide chain of the protein. The results show that the enoyl reductase lies between the 3-oxoacylsynthase and the acyl carrier. This confirms that the active sites occur in a different order on the single multifunctional polypeptide of vertebrate fatty acid synthase and the two multifunctional polypeptides of fungal fatty acid synthase, and suggests that these two systems have arisen by independent gene fusion events.

Animals↗
PubMed1983

Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase.

Rabbit mammary fatty acid synthase was labelled in the acyl transferase domain(s) by the formation of the O-ester intermediates after incubation with [14C]acetyl- or malonyl-CoA. Elastase peptides containing the labelled acyl groups were isolated using high performance liquid chromatography and sequenced by fast atom bombardment mass spectrometry. An identical peptide (acyl-Ser-Leu-Gly-Glu-Val-Ala) was obtained after labelling with acetyl- or malonyl-CoA. This confirms the hypothesis that, unlike Escherichia coli or yeast, a single transferase catalyses the transfer of both acetyl- and malonyl-groups in the mammalian complex. The sequence at this site is compared with that around the active serine in other acyl transferases and hydrolases.

Acyltransferases↗
PubMed1983

The multifunctional polypeptide chains of rabbit-mammary fatty-acid synthase. Stoichiometry of active sites and active-site mapping using limited proteolysis.

Several methods have been used to label active centres on the multifunctional polypeptide chains of rabbit mammary fatty acid synthase. Experiments using [14C]acetyl-CoA or [14C]malonyl-CoA have shown that there is a single non-thiol site which binds either acetyl or malonyl groups, present at a stoichiometry of two per enzyme dimer, and representing an intermediate in the acyl transferase reaction. This adds further support to the view that the two subunits are identical and that each polypeptide chain contains up to seven active centres. However, two novel and independent methods for the quantification of the pantetheine thiol demonstrate that this prosthetic group can be present in sub-stoichiometric amounts. By studying intermediates during limited elastase digestion of fatty acid synthase labelled in different active centres, we have been able to map the positions of four active centres within the polypeptide chain. The thioesterase domain is present in a terminal location on both polypeptide chains as previously reported. The acyl carrier domain (pantetheine thiol) is located in a region of molecular weight 9000 immediately adjacent to the thioesterase domain. The acyl transferase (acyl-O-ester site) and the 3-oxoacylsynthase thiol are located in a region of molecular weight 110000 at the opposite end of the polypeptide chain to the thioesterase domain. The relationship between the disposition of the activities on the multifunctional polypeptide chains of yeast and mammalian fatty acid synthase is discussed.

Acetyl Coenzyme A↗
PubMed1982

Evidence that the acyl-O-esters are intermediates in the catalysis. The mechanism of rabbit mammary fatty acid synthase.

The sequence acetyl-CoA leads to acetyl-O-enzyme leads to acetyl-S-acyl carrier protein has for the first time been demonstrated directly with a multifunctional (mammalian) fatty acid synthase. This was achieved by blocking of the active-site thiols of rabbit mammary fatty acid synthase with iodoacetamide. The modified enzyme was incubated with [14C]acetyl-CoA to form acetyl-O-enzyme, and acetyl-CoA was removed rapidly by centrifuge desalting. We were then able to demonstrate transfer of the acetyl group from [14C]acetyl-O-enzyme to the pantetheine thiol in a fragment of rabbit mammary fatty acid synthase containing the phosphopantetheine group, and to E. coli acyl carrier protein.

Acetyl Coenzyme A↗
PubMed1982

Reaction of chloroacetyl-CoA with rabbit fatty acid synthase. A new method to label specifically and quantify pantetheine prosthetic groups.

The substrate analogue chloroacetyl-CoA inhibits fatty acid synthase by reacting with the 'central' or pantetheine thiol and not the 'peripheral' or beta-ketoacylsynthase thiol as previously reported. This was demonstrated by the isolation of [14C]carboxymethylcysteamine after acid hydrolysis of enzyme labelled with chloro[14C]acetyl-CoA, and by the demonstration that more than one of the partial reactions is inhibited. This reagent now represents a simple and convenient tool both for quantification of the pantetheine thiol and for labelling this site for peptide mapping and isolation.

Acetyl Coenzyme A↗
PubMed1981

Sedimentation properties of native and proteolysed preparations of ox glutamate dehydrogenase.

The concentration-dependent aggregation behaviour of purified ox liver and brain glutamate dehydrogenase preparations was compared with that of commercially-obtained preparations of the liver enzyme, which have recently been shown to have suffered proteolytic cleavage. Although there were no significant differences in these effects, the presence of 3 mM-GTP and 3 mM-NADH had markedly different effects on the two types of preparation. In this situation, at higher protein concentrations the commercially obtained preparations existed in a higher degree of aggregation than those which had not suffered proteolysis. Studies of the effects of GTP and NADH concentrations on the sedimentation coefficients at a fixed enzyme concentration suggested these effects to be largely due to differences in the affinities of the two preparations for nucleotides.

Animals↗
PubMed1980

Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage.

1. A rapid procedure, involving ion-exchange chromatography on DEAE-cellulose and affinity chromatography on GTP-Sepharose, was used to purify glutamate dehydrogenase from ox brain and liver. 2. Preparations purified in this way differed from those of the ox liver enzyme that were obtained from commercial suppliers in their mobilities on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. This difference appears to result from the occurrence of limited proteolysis during the preparation of the latter enzyme samples. 3. N-Terminal sequence analysis showed the presence of four amino acid residues in the enzyme prepared in this study that were not present in those obtained from the commercial sources and which have not been reported in previous studies on the sequence of the ox liver enzyme. 4. A preliminary examination of the enzyme prepared in this way indicated that the Michaelis constants for the substrates are similar to those obtained from the commercial preparation, but that the response to allosteric effectors was modified.

Amino Acid Sequence↗