Conversion of canavanine to alpha-keto-gamma-guanidinooxybutyrate and to vinylglyoxylate and 2-hydroxyguanidine.

It was observed previously that hydroxyguanidine is formed in the reaction of canavanine(2-amino-4-guanidinooxybutanoate) with amino acid oxidases. The present work shows that hydroxyguanidine is formed by a nonenzymatic beta,gamma-elimination reaction following enzymatic oxidation at the alpha-C and that the abstraction of the beta-H is general-base catalyzed. The elimination reaction requires the presence in the alpha-position of an anion-stabilizing group--the protonated imino group (iminium ion group) or the carbonyl group. The iminium ion group is more activating than the carbonyl group. Elimination is further facilitated by protonation of the guanidinooxy group. The other product formed in the elimination reaction was identified as vinylglyoxylate (2-oxo-3-butenoate), a very highly electrophilic substance. The product resulting from hydrolysis following oxidation was identified as alpha-keto-gamma-guanidinooxybutyrate (ketocanavanine). The ratio of hydroxyguanidine to ketocanavanine depended upon the concentration and degree of basicity of the basic catalyst and on pH. In the presence of semicarbazide, the elimination reaction was prevented because the imino group in the semicarbazone derivative of ketocanavanine is not significantly protonated. Incubation of canavanine with 5'-deoxypyridoxal also yielded hydroxyguanidine. Since the elimination reactions take place under mild conditions, they may occur in vivo following oxidation at the alpha-C of L-canavanine (ingested or formed endogenously) or of other amino acids with a good leaving group in the gamma-position (e.g., S-adenosylmethionine, methionine sulfoximine, homocyst(e)ine, or cysteine-homocysteine mixed disulfide) by an L-amino acid oxidase, a transaminase, or a dehydrogenase. Therefore, vinylglyoxylate may be a normal metabolite in mammals which at elevated concentrations may contribute to the in vivo toxicity of canavanine and of some of the other above-mentioned amino acids.

Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosophila melanogaster: a putative juvenile hormone in the higher Diptera.

The in vitro production of juvenile hormone (JH) was investigated by using isolated ring glands from third instar Drosophila melanogaster. A JH-like molecule is secreted that comigrates with a synthetic sample of methyl 6,7;10,11-bisepoxy-3,7,11-trimethyl-(2E)-dodecenoate (JHB3) during TLC, liquid chromatography, and GC analysis. Purified product from farnesoic acid-stimulated ring glands was analyzed by electron impact GC/MS and gave a mass spectrum identical to synthetic JHB3. Additional structure confirmation was obtained following conversion of product from unstimulated biosynthesis to a derivative that comigrated on liquid chromatography with the derivative prepared from synthetic JHB3. Physiological studies revealed that JHB3 is produced solely by the corpus allatum portion of the ring gland in vitro. Isolated ring glands from other cyclorrhaphous dipteran larvae also produce JHB3 almost exclusively in vitro. Corpora allata from mosquito larvae, however, produce only JH III, indicating that JHB3 production may be restricted to the higher Diptera. Topically applied synthetic JHB3 caused developmental responses in newly formed D. melanogaster white puparia similar to those obtained with JH III. The data suggest that JHB3 is a fly juvenile hormone.

Activation of synthesis of hexacosenoic acid by sulfhydryl reagents in swine cerebral microsomes.

The elongation of icosenoyl-CoA (20:1-CoA) in swine cerebral microsomes resulted in the synthesis of docosenoic acid (22:1) and tetracosenoic acid (24:1), but the synthesis of hexacosenoic acid (26:1) was negligible. In contrast, in the presence of sulfhydryl reagents (0.6 mM N-ethylmaleimide [NEM] or 0.3 mM p-chloromercuriphenylsulfonic acid [PCMPS]) the synthesis of 26:1 was remarkably enhanced. We suggest that the synthesis of 26:1 from 20:1-CoA was more enhanced by NEM or PCMPS as a result of activation of the condensation step in the elongation of 24:1 (intermediate) to 26:1.

Lipid biosynthesis and composition in oil bodies and microsomes of olive fruit.

Both lipid synthesis and composition in oil bodies and microsomes of olive fruit at the first stage of development have been studied. The rate of fatty-acid synthesis in isolated oil bodies was saturated by 4.0 microM [2-14C]-malonyl-CoA. The fatty-acids synthesized of phospholipids and neutral lipids were saturated and monounsaturated. Neutral lipids, galactolipids and, above all, phospholipids were the major acyl-lipid components of microsomal fraction, oleic and palmitic being their principal fatty-acids. When the lipids of microsomes were labelled in vivo with [1-14C]-acetate, phospholipids and neutral lipids exhibited a higher biosynthesis rate relative to the galactolipids. The increase in saturated and monounsaturated fatty-acid synthesis in microsomes, was also accompanied by an important [1-14C]-acetate incorporation into polyunsaturated acids. The data presented here, in conjunction with our previous morphological results, suggest the possibility that olive fruit oil bodies could contain the necessary enzymes for the reserve lipid biosynthesis.