Biosynthesis of prostaglandins

Highly purified cyclooxygenase from sheep vesicular glands is stimulated by the presence of protoporphyrin IX compounds. This stimulation may be due to the conversion of an apoenzyme to the holoenzyme, and full activity is achieved when half of the enzyme subunits (70,000 daltons) bind heme. Also, one‐half of the subunits appear to contain non‐heme iron. The apparent molecular weight of the holoenzyme is approximately 300,000 daltons and is compatible with a complex of four 70,000 dalton subunits. Thus, we suggest that heme and non‐heme iron may be attached to different 70,000 daltons subunits that make up an A2B2‐type of peptide chain arrangement.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141216/1/lipd0591.pd

Phospholipid reactivation of plasmalogen metabolism

This report is concerned mainly with the properties of an enzyme from rat liver microsomes which hydrolyzes the alkenyl ether bond of 1‐(1′‐alk‐1′‐enyl)‐glycero‐3‐phosphoryl‐choline (alkenyl‐GPC hydrolase).Destruction of the normal environment of the microsomes by treatment with phospholipases A or C caused inactivation of the alkenyl‐GPC hydrolase, which was then partially reactivated by the addition of exogenous phospholipids. Both sphingomyelin and diacyl‐GPC were efficient in restoring activity; diacyl‐GPE was less effective; and monoacyl‐GPC and monoacyl‐GPE were ineffective. The presence of two long hydrocarbon chains in the lipid activator is apparently required for reactivation, suggesting that interaction of hydrophobic areas of the enzyme with the phospholipid is necessary for maximal activity. High concentrations of sucrose mimicked the effect of phospholipids, and because the sucrose and diacyl‐GPC did not show an additive effect, they may reactivate the enzyme in a similar manner.Disrupting the enzyme’s environment by freezing and thawing the preparation also resulted in a loss of enzymatic activity, which was restored by added exogenous phospholipids.The alkenyl‐GPC hydrolase was inhibited by imidazole and some of its derivatives. Histidine and N‐acetyl histidine did not inhibit the enzyme, presumably due to the presence of a negative charge on the carboxyl group rather than the steric bulk of that group, since histidine methyl ester did inhibit the enzyme. Kinetic evidence showed imidazole to be a competitive inhibitor. The enzymatic activity of imidazole‐treated microsomes also increased following addition of exogenous phospholipids. Imidazole inhibition differed from the phospholipase A‐inactivation in that it was partially reversed by KCl, but not by sucrose. Imidazole did not inhibit other microsomal enzymes tested, indicating that it is not a general inhibitor of membrane‐associated enzymes.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141883/1/lipd0111.pd

A solved problem of triglyceride analysis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141434/1/lipd0295.pd

FACTORS REGULATING THE BIOSYNTHESIS OF VARIOUS PROSTAGLANDINS

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74636/1/j.1749-6632.1971.tb53190.x.pd

Formation of acyl and alkenyl glycerol derivatives in Clostridium butyricum

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32807/1/0000180.pd

Metabolism of plasmalogen III. Relative reactivities of acyl and alkenyl derivatives of glycerol-3-phosphorylcholine

1. (1) The alkenyl ether derivatives of phospholipids (plasmalogens) react at slower rates than the acyl analogs in several enzyme-catalyzed reactions.2. (2) Alkenylglycerol-3-phosphorylcholine is essentially inert as a substrate for acyl-CoA: phospholipid acyltransf erase. This result suggests that in vivo the 2-acyl substituent may be present before the alkenyl ether group is formed in the molecule.3. (3) Alkenyl acylglycerol 3-phosphorylcholine is essentially inert as a substrate for cabbage phospholipase D (EC 3.1.4.4.). This lack of reactivity allows a convenient separation of alkenyl acylglycerol 3-phosphorylcholine from its diacyl analog in naturally occurring mixtures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32018/1/0000060.pd

Phospholipid precursors of prostaglandins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33093/1/0000479.pd

A test for the dihydroxyacetone phosphate pathway

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32658/1/0000023.pd

Selective effects of fatty acids upon cell growth and metabolic regulation

Positional isomers ofcis‐methyleneoctadecanoic acid differed greatly in their efficiency for growth of an unsaturated fatty acid auxotroph ofEscherichia coli upon glucose as a carbon source. The 8, 9, and 11 isomers were more efficient in producing cells (60–70 cells/fmole) than the others (0–7 cells/fmole), although all isomers were found esterified to a similar extent into cellular lipid. WithSaccharomyces cerevisiae mutants, all isomers between 6 and 12 supported some growth of the eukaryotic cells, and the 7 and 9 isomers were slightly more efficient than the 8‐isomer. WhenE. coli were grown with glycerol, all isomers from 5 to 14 supported growth, and those with the substituent near the center of the acyl chain had the greatest efficiency (70 cells/fmole). With the glycerol medium, the pattern of efficiencies for the variouscis‐methylene acyl chains resembled the broad selectivity reported earlier for thecis‐ethylenic isomers in glucose medium, which agreed closely with predictions based upon the physical property of their phospholipid derivatives. Thus, metabolism of glycerol appeared to allow the cyclopropane acyl chains to support cell functions to the limits expected for bulk phase chain‐chain fluidity considerations. This broad specificity was also obtained when cells were grown on glucose with cyclic AMP added to the culture. Therefore, the selective inadequacies of the 5, 6, 7, 10, 12 and 13 isomers in supporting cell growth on glucose may occur through an interaction modified by cAMP and dependent upon reduced cellular levels of cyclic AMP. The highly selective pattern of efficiency of thecis‐methylene acids forE. coli growth on glucose resembles that with the acetylenic acids, but was shifted one carbon atom toward the methyl terminus. This observed selectivity pattern seems due to interactions of the individual acyl chains with cellular protein(s) rather than to chain‐chain interactions in a bulk phase. The ability of certain positional isomers to support cell function equally well in both nutrient conditions suggests that the role of those acyl chain isomers may be independent of metabolite flux or cyclic nucleotide contents of the cell, whereas the actions of other isomeric fatty acids seem closely related to the metabolic status of the cell. A highly selective role for different fatty acids in modulating cellular function seems possible on the basis of the current evidence.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141577/1/lipd0878.pd

Evidence for an activating factor formed during prostaglandin biosynthesis

SummaryThe oxygenation of 5,8,11,14-eicosatetraenoic acid by acetone powder preparations of sheep vesicular glands proceeded with a lag or accelerative phase when inhibitory concentrations of NaCN were present. This accelerative feature of the reaction suggested that an activating material might be produced as the oxygenation reaction proceeds. When a second addition of fresh enzyme was made to a reaction mixture, the lag phase was as short as in uninhibited controls. This indicated that an activating factor was required for optimal activity of this dioxygenase, and that it accumulated during the oxygenation reaction in the presence of NaCN. The factor was extracted from an aqueous incubation medium with cold diethyl ether. There was a positive relationship between the amount of activating factor added and the resultant increase in the initial velocity of the oxygenation system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22023/1/0000439.pd