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A J Fulco

Publications and source records attributed to A J Fulco.

At least 55 records · Page 3Linked to original sources

A rapidly metabolizing pool of phosphatidylglycerol as a precursor of phosphatidylethanolamine and diglyceride in Bacillus megaterium.

Pulse-chase experiments in Bacillus megaterium ATCC 14581 with [U-14C]palmitate, L-[U-14C]serine, and [U-14C]glycerol showed that a large pool of phosphatidylglycerol (PG) which exhibited rapid turnover in the phosphate moiety (PGt) underwent very rapid interconversion with the large diglyceride (DG) pool. Kinetics of DG labeling indicated that the fatty acyl and diacylated glycerol moieties of PGt were also utilized as precursors for net DG formation. The [U-14C]glycerol pulse-chase results also confirmed the presence of a second, metabolically stable pool of PG (PGs), which was deduced from [32P]phosphate studies. The other major phospholipid, phosphatidylethanolamine (PE), exhibited pronounced lags relative to PG and DG in 14C-fatty acid, [14C]glycerol, and [32P]phosphate incorporation, but not for incorporation of L-[U-14C]serine into the ethanolamine group of PE or into the serine moiety of the small phosphatidylserine (PS) pool. Furthermore, initial rates of L-[U-14C]serine incorporation into the serine and ethanolamine moieties of PS and PE were unaffected by cerulenin. The results provided compelling in vivo evidence that de novo PGt, PS, and PE synthesis in this organism proceed for the most part sequentially in the order PGt yields PS yields PE rather than via branching pathways from a common intermediate and that the phosphatidyl moiety in PS and PE is derived largely from the corresponding moiety in PGt, whereas the DG pool indirectly provides an additional source for this conversion by way of the facile PGt in equilibrium or formed from DG interconversion.

Bacillus megaterium↗

Characteristics of a cytochrome P-450-dependent fatty acid omega-2 hydroxylase from bacillus megaterium.

The fatty acid (omega-2) hydroxylase from Bacillus megaterium ATCC 14581 was examined with respect to some general enzymatic properties attributed to an intact complex isolated in a partially purified state. Hydroxylase specific activity was found to increase with increasing protein concentration in a manner consistent with a reversible association of the components in the complex. There was a substantial kinetic lag phase for palmitate hydroxylation which was abolished by a substrate preincubation in the absence of NADPH. The substrate bound and presumably activated the hydroxylase complex without the formation of a substrate-derived intermediated. The oxidation of NADPH and the hydroxylation of palmitate were found to occur in a one to one molar ration, independent of the protein concentration. Finally, a cytochrome P-450 component of the complex was identified on the basis of its CO-binding difference spectrum. It appears, that this cytochrome P-450 component is not identical to P-450 meg of the steroid hydroxylase system of B. megaterium ATCC 13368, since progesterone, an active substrate for the latter, is not hydroxylated by the preparation from B. megaterium ATCC 14581.

Bacillus megaterium↗

Involvement of a single hydroxylase species in the hydroxylation of palmitate at the omega-1, omega-2 and omega-3 positions by a preparation from Bacillus megaterium.

A soluble enzyme preparation from Bacillus megaterium, requiring NADPH and O2 for activity and containing ferredoxin-replaceable and cytochrome P-450-type components, was previously shown to catalyze the conversion of palmitic acid to an isomeric mixture of omega-1, omega-2 and omega-3 hydroxypalmitate. It has now been shown that the ratio of these three positional isomers in the enzymatic product remains unchanged in spite of partial diminution of total hydroxylase activity by heat treatment, pH change or inhibition by p-hydroxy-mercuribenzoate or carbon monoxide. These findings strongly support the hypothesis that a single hydroxylase with one substrate binding site is responsible for hydroxylation at all three positions of palmitate.

Bacillus megaterium↗

Omega-1, Omega-2 and Omega-3 hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megaterium.

A soluble enzyme preparation from Bacillus megaterium, previously shown to hydroxylate free fatty acids to isomeric mixtures of Omega-1, Omega-2 and Omega-3 monohydroxy fatty acids in the presence of NADPH and O2, has now been shown to act also on fatty amides but not only hydrocarbons or fatty acid methyl esters. Using 14-C-labelled substrates, both the chain-length specificity and the positional specificity of hydroxylation was determined for fatty acids, alcohols and amides. The most active saturated fatty acid (pentadecanoic) was hydroxylated at a rate 10 times greater than the most active amide (myristamide) and 14 times faster than the most active alcohol (1-tetradecanol). Among the saturated fatty acids, the order of activity as hydroxylation substrates was C15 greater than C16 greater than C14 greater than C17 greater than C13 greater than C18 = C12. For amides the order was C14 greater than C12 greater than C15 greater than C16 while for alcohols it was C14 greater than C13 = C15 greater than C12 greater than C15. Four cis-monounsaturated fatty acids were also tested. Oleic, palmitoleic and cis-12-octadecenoic acids were more active than their saturated analogs but cis-5-tetradecenoate was less active than myristate. For all of the substrates mentioned above, with the possible exception of several unsaturated acids, the alkyl chains were monohydroxylated to give isomeric mixtures of the Omega-1, Omega-2 and Omega-3 derivatives. The distribution of these three isomers varied with chain-length and type of substrate but generally, the Omega-2 position was favored. The terminal methyl (Omega) group of these substrates was never hydroxylated and there did not appear to be significant hydroxylation of methylene carbons beyond the Omega-3 position. Based on the data presented here and in a previous paper, a model is proposed for the enzyme-substrate complex which involves hydrophobic binding and sequestering of the terminal methyl group of the substrate and electrostatic binding of the substrate's polar functional group.

Amides↗