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Biomedical subjects

D R Morris

Publications and source records attributed to D R Morris.

At least 91 records · Page 5Linked to original sources
PubMed1978

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.

Adenosylmethionine Decarboxylase↗
PubMed1978

Some factors affecting erythrocyte deformability and rupture.

Human erythrocytes suspended in a viscous isotonic saline medium may be ruptured in vitro by the application of uniform hydrodynamic shear stresses. For any given combination of exposure parameters, cellular fragility is a measure of cellular deformability; i.e., it is related to the cellular dimensions that result from the competition between those forces that tend to extend the cell and those forces--particularly those associated with the resistance to bending of the membrane--that tend to restore the cell to its original biconcave shape. The pharmacologically active agent nicotinic acid increases cellular fragility in our experimental system and so has presumably increased the deformability of the human erythrocyte.

Blood Viscosity↗
PubMed1977

Comparison of the biosynthetic and biodegradative ornithine decarboxylases of Escherichia coli.

Biosynthetic ornithine decarboxylase was purified 4300-fold from Escherichia coli to a purity of approximately 85% as judged by polyacrylamide gel electrophoresis. The enzyme showed hyperbolic kinetics with a Km of 5.6 mM for ornithine and 1.0 micronM for pyridoxal phosphate and it was competitively inhibited by putrescine and spermidine. The biosynthetic decarboxylase was compared with the biodegradative ornithine decarboxylase [Applebaum, D., et al. (1975), Biochemistry 14, 3675]. Both enzymes were dimers of 80 000-82 000 molecular weight and exhibited similar kinetic properties. However, they differed significantly in other respects. The pH optimum of the biosynthetic enzyme was 8.1, compared with 6.9 for the biodegradative. Both enzymes were activated by nucleotides, but with different specificity. Antibody to the purified biodegradative ornithine decarboxylase did not cross-react with the biosynthetic enzyme. The evolutionary relationship of these two decarboxylases to the other amino acid decarboxylases of E. coli is discussed.

Carboxy-Lyases↗
PubMed1977

Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.

Animals↗
PubMed1975

Biodegradative ornithine decarboxylase of Escherichia coli. Purification, properties, and pyridoxal 5'-phosphate binding site.

The biodegradative ornithine decarboxylase of Escherichia coli has been purified to apparent homogeneity. At its pH optimum (pH 7.0), the enzyme exists as a dimer of 160,000 molecular weight. Aggregation of the dimer was promoted by lower pH values. The enzyme requires pyridoxal 5'-phosphate for activity. The coenzyme appears to be bound in Schiff base linkage as suggested by spectral studies and inhibition by NaBH4. The following sequence was determined for the coenzyme binding site: Val-His-(epsilon-Pxy)Lys-Gln-Gln-Ala-Gly-Gln. The properties of this enzyme are compared with the other biodegradative amino acid decarboxylases that have been isolated from E. coli.

Amino Acid Sequence↗
PubMed1975

Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.

There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication.

Animals↗
PubMed1975

Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.

Electrophoresis of ribosomal RNA in polyacrylamide-agrose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel elctrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.

Barium↗
PubMed1974

Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.

Electrophoresis of ribosomal RNA in polyacrylamide-agarose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel electrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.

Barium↗
PubMed1974

Polyamine limitation of growth slows the rate of polypeptide chain elongation in Escherichia coli.

The rate of polypeptide chain elongation during steady-state, polyamine-limited growth of a mutant of Escherichia coli was measured by two independent techniques. Analysis of polysome patterns gave values of 17.5 and 9.5 amino acids per s at 37 C in unstarved and polyamine-limited cells, respectively. From the kinetics of entry of labeled amino acids into polypeptides of defined molecular weights, values at 30 C of 10.1 and 5.8 amino acids per s were obtained for unstarved and polyamine-limited cultures, respectively. Correction of these values to 37 C resulted in rates of 15.0 and 8.7 amino acids per s. These results support the previous conclusion, based on the kinetics of beta-galactosidase induction, that polyamine starvation decreases the rate of protein synthesis by limiting the velocity of polypeptide chain elongation.

Bacterial Proteins↗