Lemuel D. Wright

A comparative study of sterol biosynthesis in Annelida

1. n1. The metabolism acid has been studied in marine and terrestrial species from the Phylum Annelida with 14C substrates. n n2. n2. Radioactive 3-β-hydroxy sterols were isolated from all polychaetes injected with 2-14C mevalonic acid; one of the labeled sterols found in Nereis diversicolor has been isolated and identified as cholesterol. n n3. n3. In contrast, no 14C-3-β-hydroxy sterols were detected in the terrestrial species, Lumbricus terrestris, but a large amount of radioactivity was found in squalene; this species can synthesize squalene, but is unable to cyclize this intermediate to digitonin-precipitable sterols. n n4. n4. Similar results were obtained both in the presence and absence of antibiotics.

Liquid scintiliation counting of radioautograms

Counting procedures are described for the liquid scintillation counting of paper sections from radioautograms. (C.H.)

Structure of Mn2+ and Cu2+ complexes with l-methionine, S-methyl-l-cysteine, l-threonine and l-serine

Abstract A comparison of the stability constants determined by several authors for complexes between metal ion, Mn 2+ through Zn 2+ , and potential tridentate amino acids, methionine, S -methylcysteine, threonine or serine, with those of the bidentate ligands, alanine or α-amino- n -butyric acid, showed that no significant increase in stability for the complexes with the potential tridentate ligands occurred. This does not necessarily suggest that an interaction between metal ions and the thioether or hydroxy groups, which are possible binding sites in these amino acids, is nonexistent. By means of nuclear magnetic resonance spectroscopy, Mn 2+ and Cu 2+ complexes with the previously mentioned amino acids were investigated. Strong evidence has suggested that the sulfur atom of S- methyl- l -cysteine is involved in complex formation with Cu 2+ , while such interaction is either small or nonexistent with Mn 2+ . Neither metal ion interacted significantly with the sulfur of l -methionine. The hydroxy group of l -threonine was involved with the binding to Mn 2+ , while no interaction seemd to occur with Cu 2+ ; this behavior was probably due to the different preferred coordination numbers of these metal ions. The proton signals of l -serine resulted in a complex pattern; therefore, no detailed conclusions could be drawn for this ligand, although it was assumed that the structures of the complexes were similar to those for l -threonine. Structures for the investigated complexes are suggested, and the significance of weak interactions in metal ion-enzyme complexes is discussed.

Effect of Nicotinic Acid and Related Compounds on Incorporation of Mevalonic Acid into Cholesterol

Summary Nicotinic acid and related compounds (0.1 to 5 mg per ml) have no significant effect on conversion of mevalonic acid to non-saponifiable material by rat liver homogenates. The incorporation of acetate into non-saponifiable material (largely cholesterol) decreases with increasing amounts of nicotinic acid. At 1 mg per ml of nicotinic acid a 34% average decrease in biosynthesis was observed. The results suggest that the locus of the nicotinic acid effect may be somewhere between acetate and mevalonate.

The Metabolism of Biotin and Analogues

Publisher Summary Biotin is required by animals and some microorganisms in extremely small amounts. Biotin is determinable in physiological amounts only by microbiological methods and it is synthesized by many microorganisms including those of the intestinal tract so that the production of a deficiency in humans and other animals is complicated, and it is covalently bound to apoenzymes rendering resolution of holoenzymes virtually impossible. The role of pimelic acid as a growth factor seems restricted to that of a precursor of biotin. Pimelic acid has been reported to be a vitamin for the larvae of the confused flour beetle, Tribolium confusum , but the activity recorded may be due to conversion to biotin by the flora of the intestinal tract. Pimelic acid fails to satisfy the biotin requirement of at least Lactobacillus casei , Lactobacillus arabinosus , biotin-requiring fungi, yeast, or Rhizobium trifolii , a very significant study of biotin biosynthesis in Achromobacter sp., which was isolated from the bovine rumen. The organism is rather anomalous because it fails to grow on media containing glucose as the main energy source, and it does not synthesize increased amounts of biotin when the medium is supplemented with pimelic acid.

Factors influencing incorporation of mevalonic acid into cholesterol by rat liver homogenates.

Summary Incorporation of mevalonic acid into cholesterol by rat liver homogenates is favored by (a) use of tissues from very young rats, (b) use of a “loose”homogenizer, and (c) saturation of the homogenate with straight oxygen and stoppering. Incorporation of mevalonic acid is less in the presence of compounds that may be intermediates or antimetabolites of cholesterol biosynthesis. With whole homogenates biosynthesis is not markedly influenced by the method of killing the rats or by additions of ATP.

Metabolism, in the Rat, of Biotin Injected Intraperitoneally as the Avidin–Biotin Complex

Summary Radioactive biotin injected intraperitoneally into rats as the avidin complex was found to be excreted much more slowly than the free vitamin. However, the biotin-avidin complex is dissociated in vivo and the released biotin both excreted and metabolized to sulfoxides and bisnorbiotin as is free biotin. That liver has the capacity to cause dissociation of the avidin-biotin complex, as well as metabolize the vitamin, was also demonstrated.

Urinary excretion of biotin and metabolites in the rat.

Summary Approximately 95% of an ip dose of 0.5 mg/100 g body weight of [14C] biotin is excreted by the young, male rat within 24 hr; 84% is excreted within the first 3 hr. The rate and extent of excretion of bisnorbiotin is essentially the same. The more water-soluble tetranorbiotin, both d-and l-sulfoxides of biotin, and biotin sulfone are excreted even more rapidly, with 96-98% of the injected dose appearing in the urine within the first few hours.

HEAT STABILITY OF AVIDIN AND AVIDIN-BIOTIN COMPLEX AND INFLUENCE OF IONIC STRENGTH ON AFFINITY OF AVIDIN FOR BIOTIN.

Summary An assay method involving the use of radioactive biotin and Sephadex chromatography was used to determine avidin and/or the avidin-biotin complex. It was demonstrated that avidin is relatively heat labile while the avidin-biotin complex is relatively heat stable. It was found that the affinity of avidin for biotin, essentially zero in aqueous solution, is a direct function of the ionic strength of the medium in which avidin and biotin react.

The incorporation of pimelic acid as a unit in the biosynthesis of biotin

Abstract Previous investigators have obtained indirect evidence that pimelic acid is a precursor of biotin (1,2,3,4,5). We have obtained direct evidence that pimelic acid serves as a precursor of biotin by isolating labeled biotin sulfone from hydrogen peroxide-treated eluates derived from culture filtrates of Aspergillus niger grown on a medium containing pimelic acid-1,7-C 14 (6). The incorporation of pimelic acid into biotin was substantiated in a later report by Eisenberg in which labeled desthiobiotin was obtained after growth of Phycomyces blakesleeanus on a medium containing labeled pimelic acid-1,7-C 14 (7). The experiments described in this paper provide evidence that pimelic acid is incorporated as a unit in the biosynthesis of biotin and offer no substantiation for the results of Eisenberg indicating a multiple entry of pimelic acid into the biotin molecule.