Date of Award

6-1-1969

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

John A. Duerre

Abstract

Radioactively labeled S-adenosyl-L-homocysteine was administered intravenously to rats to determine the fate of the homocysteine moiety and to isolate and identify any excretory products that arose from the metabolism of this compound in vivo. After intravenous injection of S-adenosyl-L-homocysteine-3H (homocysteine labeled) into rats, less than 15% of the radioisotope was incorporated into protein methionine or excreted as α-ketobutyrate. The remaining tritium was associated with a previously unidentified keto acid which was excreted in the urine. Intravenous injections of S-adenosyl-3H-L-homocysteine (adenosine labeled) or S-adenosyl-L-homocysteine-35S revealed that both the purine moiety and the sulfur atom remained associated with the keto acid excretory product. The compound was isolated from the urine by ion-exchange chromatography, purified and crystallized. Based on chemical, elemental, and ultraviolet and infrared spectral analyses as well as the information obtained from the radioisotope tracer studies, the chemical structure of the compound was proposed as S-adenosyl-γ-thio-α-ketobutyrate.

To determine the type of reaction responsible for the formation of S-adenosyl-γ-thio-α-ketobutyrate, S-adenosyl-3H-L-homocysteine was incubated with various rat tissue extracts under conditions which would favor oxidative deamination or transamination. Radioactive S-adenosyl-γ-thio-α-ketobutyrate was isolated by chromatography from reaction mixtures in which kidney and liver extracts were used. The reaction was found to be catalyzed by the general L-amino acid oxidase, EC 1.4.3.2. Incubation of the partially purified oxidase with S-adenosyl-L-homocysteine resulted in the oxidative deamination of the substrate to S-adenosyl-γ-thio-α-ketobutyrate with the utilization f 0.48 μmoles of oxygen per μmole of ammonia and keto acid formed in the presence of catalase. Without catalase oxygen consumption was doubled. The pH optimum for S-adenosyl-γ-thio-α-ketobutyrate formation ranged from 8.8 to 9.2 and the Km value for S-adenosyl-L-homocysteine was 2.5 x 10-2 M as compared to those of 1.3 x 10-2 M and 1.9 x 10-2 M for L-leucine and L-methionine, respectively.

Cell-free extracts of the major rat tissues were surveyed for the presence of S-adenosyl-L-homocysteine hydrolase, and only liver extracts exhibited activity. When isolated livers were perfused with buffer containing erythrocytes, serum albumin and tritiated S-adenosyl-L-homocysteine, neither hydrolysis to adenosine and L-homocysteine not deamination to of S-adenosyl-γ-thio-α-ketobutyrate occurred. The apparent impermeability of the live cells to S-adenosyl-L-homocysteine would explain why this compound was not readily hydrolyzed during the in vivo experiments. However, formation of S-adenosyl-L-homocysteine intracellularly in the liver as a result of transmethylation reactions, could be subsequently followed by hydrolysis to adenosine and L-homocysteine. S-adenosyl-L-homocysteine formed in other organs would not be hydrolytically cleaved but could be oxidatively deaminated by the kidney L-amino acid oxidase and eliminated in the urine as of S-adenosyl-γ-thio-α-ketobutyrate.

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