S.S. Kerwar

Studies on the initiation of protein synthesis in animal tissues

Two methionine accepting tRNAs have been separated from rabbit liver tRNA by column chromatography on BD-cellulose. One of the species (Met-tRNAFMet) can be formylated using an Escherichia coli transformylase preparation, and maximal binding of this species to brain ribosomes is achieved in the presence of AUG, GTP and a 0.5 M NH4Cl extract of the ribosomes. Transferase I cannot replace the NH4Cl extract in the binding assay. The other tRNA species (Met-tRNAMet) requires GTP and transferase I for binding to ribosomes.

Studies on vitamin B12 metabolism in HeLa cells.

Abstract In the present report some aspects of vitamin B 12 metabolism in HeLa cells and other tissue culture lines have been investigated. Mangum et al . (1) previously reported the presence of higher levels of N 5 -methyl- H 4 -folate-homocysteine transmethylase in tissue culture cells grown in the presence of vitamin B 12 . The present experiments indicate that under the growth conditions used, HeLa cells do not contain sufficient vitamin B 12 to satisfy the cobamide requirement of the transmethylase, and the increase in enzymatic activity that is observed when the cells are grown in the presence of vitamin B 12 results from the conversion of the apoenzyme form of the enzyme to active holoenzyme. No effect of the vitamin on the oxidation of propionate to CO 2 was observed in these cells suggesting that the amount of 5′-deoxyadenosyl B 12 (DBCC) present was sufficient so that the methylmalonyl CoA isomerase reaction was not rate limiting. Extracts of HeLa cells readily convert hydroxy-B 12 to DBCC in the presence of ATP and a reducing system. The optimum conditions for the enzymatic synthesis of DBCC are described.

Studies on the ability of norleucine to replace methionine in the initiation of protein synthesis in E. coli

Abstract It has been previously shown that norleucine can acylate both tRNAFMet and tRNAFMet and be converted to fNorleu-tRNAFMet. The present studies have compared the reactivity of fNorleu-tRNAFMet with fMet-tRNAFMet in various reactions associated with protein synthesis. No significant differences were observed in the binding to ribosomes, reaction with puromycin, and deformylation reaction. However, unlike methionine, norleucine was not able to repress any of the enzymes involved in methionine synthesis.

Methionine biosynthesis in normal and transformed fibroblasts

Abstract SV-40 transformed human fibroblasts show a growth requirement for methionine, whereas normal fibroblasts do not. Activities of the N 5 -methyltetrahydrofolate-homocysteine transmethylase and N 5–10 -methylenetetrahydrofolate reductase in extracts of both cell lines are similar. These observations indicate that the absolute growth requirement for methionine observed in these transformed cells does not necessarily involve a deficiency in enzymes related to methionine synthesis.

The in vivo inhibition of collagen synthesis and the reduction of prolyl hydroxylase activity by 3,4-dehydroproline.

Abstract Various proline analogs and iron chelators were tested for their effect on collagen formation which occurs in the uterus of the immature rat following the administration of estradiol-17β. dl -3,4-Dehydroproline, l -α-azetidine-2-carboxylic acid and l -pyroglutamic acid reduced the estradiol-17β stimulated formation of hydroxyproline which occurs in the uterus following administration of the hormone while l -thiazolidine-4-carboxylic acid was without effect on this response. The activity of the d - and l -isomers of 3,4-dehydroproline was compared with the racemic mixture; the l -isomer was twice as active as the latter, while the d -isomer was only half as active. l -3,4-Dehydroproline was approximately four times as potent as l -α-azetidine-2-carboxylic acid, the second most active analog of those tested. dl -3,4-Dehydroproline inhibited the incorporation of l -[ 14 C]proline into the proline and hydroxyproline of uterine collagen; it also inhibited the incorporation of [ 14 C]glycine into collagen while having less effect on the incorporation of these amino acids into noncollagen protein. These results indicate dl -3,4-dehydroproline is a fairly specific and potent inhibitor of collagen formation in vivo . These observations indicate that dl -3,4-dehydroproline reduces the hydroxylation of prolyl residues in collagen. Presumably, this occurs in part due to the incorporation of the analog into the collagen molecule in place of proline. It is probably also related to a reduction of prolyl hydroxylase activity which can be demonstrated in the tissues of animals treated with 3,4-dehydroproline. A significant reduction of prolyl hydroxylase activity was shown to persist in the uterus, lung, and heart for approximately 24 h following a single intraperitoneal dose of dl -3,4-dehydroproline (200 mg/kg).

Studies on the effect of l-3,4-dehydroproline on collagen synthesis by chick embryo polysemes

Abstract Various proline analogs have been tested in vitro for their ability to inhibit the enzymatic aminoacylation of tRNA by proline. Of these, l -3,4-dehydroproline is the most potent inhibitor. This inhibition is competitive; the K i is 100 μ m . It was shown that l -3,4-dehydroproline can serve as substrate in the aminoacylation reaction. However, the incorporation of radioactivity from l -3,4-[ 14 C]dehydroprolyl-tRNA into protein occurs at one-fifth the rate observed for l -prolyl-tRNA. The addition of l -3,4-dehydroproline in vitro inhibits the synthesis of collagen to a greater extent than non-collagen protein.

Reduction of prolyl hydroxylase activity in L-929 fibroblasts by proline analogs

Abstract Various proline analogs were examined for their effect on the activity of prolyl hydroxylase in L-929 fibroblasts. Of the analogs tested, L-3,4-dehydroproline and L-azetidine-2-carboxylic acid were effective in reducing the activity of prolyl hydroxylase. A time course on the effect of these analogs showed that the reduction of enzyme activity by L-3,4-dehydroproline occurred at a faster rate than the reduction which occurs with L-azetidine-2-carboxylic acid. The results demonstrate that L-3,4-dehydroproline is approximately four times as potent as L-azetidine-2-carboxylic acid in reducing prolyl hydroxylase activity.

Further studies on the cell-free synthesis of procollagen-collagen by chick embryo polysomes

Abstract Free and membrane bound polysomes were prepared from 8-day-old chick embryos. Both polysome preparations were equally active in protein synthesis but procollagen-collagen synthesis was carried out exclusively by the membrane bound polysomes. The collagenous product was analyzed by DEAE-cellulose chromatography and after hydroxylation with peptidyl proline hydroxylase had a hydroxyproline/proline ratio of 0.77. This suggests that the collagenous product synthesized by the membrane bound polysomes is procollagen.

Studies on the nature of procollagen synthesized by chick embryo polysomes

Abstract Analysis of pro-α chains released from chick embryo membrane polysomes indicates that they are not disulfide bonded and have a molecular weight of approximately 120,000. Therefore, disulfide bonding which has been observed between pro-α chains is not essential for their completion and release.