Frank A. Lornitzo

Separation of two active forms (holo-a and holo-b) of pigeon liver fatty acid synthetase and their interconversion by phosphorylation and dephosphorylation

Summary Apo-, holo- a - and holo- b -pigeon liver fatty acid synthetases 4 were separated by affinity gel chromatography under conditions identical to those used for the separation of apo and holo forms [Qureshi et al . (1975) Biochem. Biophys. Res. Commun. 64, 836-844], except that an additional elution step at a high salt concentration, pH 7.0, and room temperature was included. The interconversion of the two forms of holo-fatty acid synthetase was then carried out in vitro . In the presence of Mg++ and a phosphatase preparation, holo- b was converted to holo- a . The reverse conversion, holo- a to holo- b , was carried out in the presence of ATP and a kinase fraction prepared from the 100, 000 g supernatant of liver homogenate. These results indicate that the interconversion of holo- a - and holo- b -fatty acid synthetases occurs by phosphorylation-dephosphorylation. These results also suggest the possibility that this process may be a mechanism of short term regulation of liver fatty acid synthetase activity.

Separation of pigeon liver apo- and holo-fatty acid synthetases by affinity chromatography☆☆☆

Abstract Apo- and holo-fatty acid synthetases of pigeon liver were separated by affinity gel chromatography under conditions similar to, but not identical to, those used in separating subunits I and II of [14C]pantetheine-labeled fatty acid synthetase complex [ Lornitzo et al. , J. Biol. Chem. 249 , 1654 (1974) ]. When [14C]pantetheine-labeled fatty acid synthetases were separated, the enzymatically active holo form contained all of the [14C] label. Incubation of the apo-pigeon liver fatty acid synthetase complex with CoA, ATP and a partially purified pigeon liver soluble enzyme system, from which fatty acid synthetase had been removed, resulted in the formation of holo-enzyme. Activation of apo-fatty acid synthetase could also be achieved by replacing the apo-(4′-phosphopantetheine-less) acyl carrier protein with holo-acyl carrier protein. It is evident, therefore, that the inactive apo-fatty acid synthetase lacks a 4′-phosphopantetheine group.

The isolation of acyl carrier protein from the pigeon liver fatty acid synthetase complex II

Abstract A low molecular weight protein of less than 10, 000 Daltons has been isolated from Subunit I (β-ketoacyl thioester reductase) of the pigeon liver fatty acid synthetase complex and purified to homogeneity. This protein contains all of the [14C]-labeled pantetheine incorporated into the fatty acid synthetase on injection of [14C]-labeled pantetheine into pigeons. It also has one β-alanine and one sulfhydryl group. This protein is an acceptor of an acetyl group from acetyl-CoA and a malonyl group from malonyl-CoA in the presence of Subunit II (transacylase). In these respects it is very similar to E. coli acyl carrier protein.

Affinity purification of anti-pigeon liver fatty acid synthetase immunoglobulin and comparative immunoreactivity of the catalytic reactions☆

Abstract Rabbit anti-pigeon liver fatty acid synthetase antibody was prepared by affinity chromatography on Sepharose-fatty acid synthetase to near monospecificity (98% or more) as shown by immunodiffusion plates and rocket immunoelectrophoresis. Immunotitrations of the highly purified monospecific antibody against the overall activity and partial activities of fatty acid synthetase were then carried out. Only 6 mol of antibody/mol of enzyme was required to inactivate overall fatty acid synthetase activity and the condensation reaction, while 12 to 18 mol were required to partially inactivate the β-ketoacyl reductase and the malonyl- and acetyl-CoA transferases. Palmitoyl-CoA thioesterase (deacylase) activity was not inhibited by the antibody. The degree of inactivation of the partial reactions by antibody was not affected by dissociation of the fatty acid synthetase. Immunoprecipitation of the enzyme indicated that there are approximately 35 immunoreactive sites on the fatty acid synthetase molecule. The possible implications of these results to an understanding of the structural organization of pigeon liver fatty acid synthetase and its antigenic determinants are discussed.

Intracellular localization of a 6-O-methyl-d-glucose containing soluble polysaccharide from Mycobacterium tuberculosis

Abstract A soluble non-dialyzable low molecular weight (4400) polysaccharide has been purified from the cell-free extract of the H37Ra strain of Myobacterium tuberculosis. The polysaccharide is not covalently bound to other cellular constituents; no hydrolytic procedures are used in its isolation. The polysaccharide is composed of d -glucose (60%) and 6-O- methyl- d -glucose (40%) and one acid group whose location is not yet known. Periodate degradation suggests that most of the 6-O- methyl- d -glucoses serve as branch points in the backbone of the polysaccharide. The polysaccharide is limited in its distribution to the soluble portion of the cytoplasm; 6-O- methyl- d -glucose is absent from hydrolysates of purified preparations of the mycobacterial cell wall, cytoplasmic membranes and ribosomes.

Apo- and holofatty acid synthetases from pigeon liver

Abstract Two forms (an apo- and a holoenzyme) of the fatty acid synthetase complex from pigeon liver were separated by affinity chromatography on a Sepharose-ϵ-aminocaproyl pantetheine column. The difference between these enzymes is the presence or absence of the prosthetic group, 4′-phosphopantetheine. Due to the absence of the prosthetic group, apofatty acid synthetase lacks the overall ability to synthesize fatty acids, and it has no β-ketoacyl synthetase (condensing enzyme) activity. These two forms of enzyme were shown to be homogeneous and they behaved identically on DEAE-cellulose chromatography, gel filtration, sucrose density gradient centrifugation, disc gel electrophoresis, and immunodiffusion. The isolation and purification of an apoacyl carrier protein are also reported. The apoacyl carrier protein lacks β-alanine and it has no sulfhydryl group, indicating therefore that the acyl carrier protein from apofatty acid synthetase does not have a 4′-phosphopantetheine group. The transfer of 4′-phosphopantetheine from CoA to apofatty acid synthetase was effected by an enzyme system present in the supernatant of pigeon liver homogenate. The resulting product of this reaction was the holofatty acid synthetase. The reverse reaction, the formation of apo- from holofatty acid synthetase, was also demonstrated. The physiological significance of this system is suggested from studies carried out on fasting and refeeding of pigeons. At early times of refeeding (0–4 h) there is a large amount of apoenzyme. The amount of holofatty acid synthetase increases after 4 h of refeeding and the apofatty acid synthetase decreases. When pigeons are refasted, after refeeding for 48 h, the amount of apoenzyme increases and the holoenzyme decreases.

A protein-bound phosphorylated product as an intermediate in the biosynthesis of O-methyl glycerol by Mycobacterium tuberculosis

Abstract 1-O-methyl sn-glycerol 3-phosphate bound to a protein has been identified as the probable intermediate in the mycobacterial transmethylation reaction leading to the formation of 1-O-methyl glycerol from S-adenosyl methionine and sn-glycerol 3-phosphate. Acid treatment of the partially purified protein-bound product appears to release 1-O-methylsn-glycerol 2:3-cyclic phosphate. Our hydrolysis studies indicate that the 1-O-methyl glycerol is bound to the protein via a phosphodiester linkage.