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Biomedical subjects

R B Trimble

Publications and source records attributed to R B Trimble.

At least 73 records · Page 4Linked to original sources
PubMed1979

Purification and properties of endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus.

An enzyme, previously described as endo-beta-N-acetylglucosaminidase L (Tarentino, A.L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817) because of its apparent specificity for Man(GlcNAc)2Asn, has been purified to homogeneity. The enzyme has now been found to hydrolyze (GlcNAc)3 to (GlcNAc)2 plus GlcNAc, and (GlcNAc)4 to 2(GlcNAc)2, at twice the rate observed for Man(GlcNAc)2Asn. Removal of the asparagine from the latter compound reduces the rate of hydrolysis by about 30-fold. Reduction of (GlcNAc)3 to GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc-ol eliminates this compound as a substrate for endo-beta-N-acetylglucosaminidase L. However, the reduction of (GlcNAc)4 does not affect its rate of hydrolysis. Endo-beta-N-acetylglucosaminidase L consists of a single polypeptide chain with a molecular weight of 49,500 +/- 400, which on isoelectric focusing separates into two closely migrating bands; a major with a pI of 4.25 and a minor one with a pI 4.20. Both bands possess similar enzyme activities and amino acid compositions, but differ slightly in their tryptic peptide maps.

Acetylglucosaminidase↗
PubMed1979

Effect of deglycosylation of yeast invertase on its uptake and digestion in rat yolk sacs.

The uptake by rat yolk sacs of native invertase and invertase which was deglycosylated by treatment with endo-beta-N-acetylglucosaminidase was compared. The initial rate of uptake of the deglycosylated enzyme was severalfold greater and its accumulation leveled off much earlier than that of the native enzyme. Uptake rates of the deglycosylated and native forms of the enzyme were proportional to their concentration in the medium in the range employed and were inhibited about 85% by 10(-6) M glucagon in both cases. After preloading of yolk sacs with native invertase, the tissue level of activity remained relatively constant over a subsequent 6-h time period, while with the deglycosylated form, activity declined substantially. Since this difference appears not to be attributable to differences in thermal stability, it is suggested that the deglycosylated form of the protein is more susceptible to intracellular proteolytic digestion. In vitro studies on the digestion of these two forms of invertase by trypsin are consistent with this suggestion.

Animals↗
PubMed1978

Identification of the transcribing DNA strand for the deoxynucleotide kinase gene of bacteriophage T2.

A modified procedure was developed which allows RNA--DNA hybridization reactions to be performed without the loss in translational capacity of mRNA which accompanies hybridization at elevated temperatures or in the presence of the denaturing agent formamide. Separated l and r strands of bacteriophage T2 DNA were hybridized in the presence of 4 M sodium perchlorate at 37 degrees C with total RNA from infected cells. After passage of the hybridization mixture through a nitrocellulose column to remove single-strand DNA and DNA--RNA hybrids, the eluent was measured for its capacity to promote deoxynucleotide kinase (gene 1) synthesis in an in vitro protein-synthesizing system derived from uninfected Escherichia coli. With this procedure, which should be of general use for any message whose product can be measured either enzymatically, immunologically, or by location in an acrylamide gel, it was demonstrated that deoxynucleotide kinase mRNA is transcribed from the l strand of bacteriophage T2 DNA. By titrating with l strand DNA, the number of deoxynucleotide kinase transcripts present 9 min after T2 phage infection at 30 degrees C was estimated to be about 38 copies per cell.

Coliphages↗
PubMed1978

Asparaginyl glycopeptides with a low mannose content are hydrolyzed by endo-beta-N-acetylglucosaminidase H.

Substrates susceptible to endo-beta-N-acetylglucosaminidase H were reduced in size through alpha-mannosidase treatment and periodate oxidation to yield the following compounds: (Man)4(GlcNAc)2Asn, [Manalpha 1 leads to 6Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNACAsn]; (Man)3(GlcNAc)2Asn, [Manalpha 1 leads to 3Man-alpha 1 leads to 6Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn]; (Man)2(GlcNAc)2Asn, [Manalpha 1 leads to 6Manbeta1 leads to 4GlcNAcbeta 1 leads to 4BlcNAcAsm]. Comparison of the relative rates of hydrolysis of these compounds with (Man)5(GlcNAc)2-Asn, the most active substrate to date for the endoglycosidase, revealed (Man)4(GlcNAc)2Asn to be hydrolyzed faster than (Man)5(GlcNAc)2Asn and (Man)3-(GlcNAc)2Asn to be equal to or slightly better than (Man)5(GlcNAc)2Asn as a substrate. (Man)2(GlcNAc)2-Asn was completely hydrolyzed but at a rate that was about 10(4) slower than (Man)5(GlcNAc)2Asn, which is comparable to that for (Man)3(GlcNAc)2Asn(aa)x [Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn(aa)x], obtained from immunoglobulin M. (Man)1(GlcNAc)2Asn, [Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn] was hydrolyzed at a 100-fold slower rate than the latter glycopeptide. The effective range of endo-beta-N-acetylglucosaminidase H has thus been extended to compounds containing as few as 2 mannosyl residues.

Acetylglucosaminidase↗
PubMed1977

Subunit structure of external invertase from Saccharomyces cerevisiae.

Because 50% of the mass of the external invertase of Saccharomyces cerevisiae consists of carbohydrate, it has been extremely difficult to obtain an accurate molecular weight of this enzyme by centrifugal or electrophoretic techniques. However, on removing almost all of the oligosaccharide chains of this enzyme with the endo-beta-N-acetyl-glucosaminidase H from Streptomyces plicatus, it has been possible to show that carbohydrate-free invertase is composed of two 60,000-dalton subunits. Terminal sequence analysis with carboxypeptidases A, B, and Y provided strong evidence that the subunits are identical.

Acetylglucosaminidase↗
PubMed1976

Level of specific prereplicative mRNA's during bacteriophage T4 regA-, 43- and T4 43- infection of Escherichia coli B.

The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP hydroxymethylase], cd [dCMP deaminase], 1 [deoxynucleotide kinase], rIIA, rIIB, 46 [DNA arrest], and 45) during extended infection of Escherichia coli B with T4 regA-, 43- and T4 43- bacteriophage. The above gene-specific transcripts in RNA isolated from infected cells were quantitated by translation with an E. coli B cell-free system. Conditions were chosen to insure that the amount of gene product formed in vitro, measured either as an enzyme activity or as a radioactive band in acrylamide gel, was directly proportional to the level of mRNA present. The failure of T4 regA-, 43- phage to terminate prereplicative synthesis (Wiberg et al., 1973) resulted in an enhanced production of many early gene products over those formed during T4 43- infection. This increase did not appear to be associated with an increment in mRNA levels, since in the present study gene-specific early mRNA's were found to be only marginally elevated and slightly more stable in T4 regA-, 43-- than in T4 43--infected cells. Of interest was the observation that significant quantities of all of the mRNA's studied; with the exception of those from genes 45 and 46, could be isolated from T4 43--infected cells after synthesis of the respective gene products had ceased. On termination of normal prereplicative synthesis during infection with T4 43- phage, polyribosomes were found to be dissociated completely, a finding which suggests that the residual mRNA present in these cells is free in the cytoplasm. The persistence in T4 43--infected cells of translatable mRNA for many prereplicative genes after product synthesis ceased indicates that the impairment in protein synthesis is not due solely to regA-mediated messenger degradation or modification. Rather, the results suggest that the regA gene product may act either by interfering with early mRNA polypeptide chain initiation or by promoting prereplicative polysome dissociation.

Cell-Free System↗
PubMed1972

The temporal expression of T2r + bacteriophage genes in vivo and in vitro.

The kinetic order of synthesis of deoxycytidylate deaminase (EC 3.5.4.12), deoxycytidylate hydroxymethylase (EC 2.1.2.b), dihydrofolate reductase (EC 1.5.1.3), 5-hydroxymethyldeoxycytidylate kinase (EC 2.7.4.4), and thymidylate synthetase (EC 2.1.1.b) after infection of Escherichia coli with T2r(+) bacteriophage was found not to correlate with their order of synthesis in an in vitro protein-synthesizing preparation. The in vivo and in vitro synthesis of enzyme-specific messenger RNA measured in the protein-synthesizing preparation preceded each enzyme by about 1 min. Through the use of sheared DNA, it was shown that the thymidylate synthetase gene was most susceptible to a loss in template activity, which suggests that this gene is further removed from its promoter than the other genes are from theirs. With a DNA segment of 2.5 x 10(5) daltons, the synthesis of dihydrofolate reductase alone was obtained, but at a much reduced rate. Translation of the RNA from phage-infected cells treated with chloramphenicol yielded amounts of dihydrofolate reductase and deoxycytidylate hydroxymethylase activities similar to those obtained with RNA from untreated infected cells. These results suggest that the chloramphenicol RNA, which consists primarily of immediate-early RNA, may contain most, if not all, of the information required for the synthesis of phage dihydrofolate reductase and deoxycytidylate hydroxymethylase.

Aminohydrolases↗
PubMed1972

Relationship between Escherichia coli B titer and the level of deoxycytidylate deaminase activity induced on bacteriophage T2r + infection.

The activities of six bacteriophage T2r(+)-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected Escherichia coli B from 8 x 10(8) to 2 x 10(8) cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 x 10(8) to 8 x 10(8) per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen.

Aminohydrolases↗
PubMed1971

Metabolism of 4-N-hydroxy-cytidine in Escherichia coli.

4-N-hydroxy-cytidine was found to substitute for uridine as a pyrimidine supplement for the growth of Escherichia coli Bu(-). Measurement of the incorporation of 4-N-hydroxy-cytidine-2-(14)C into ribonucleic acid and deoxyribonucleic acid revealed that this compound was converted to cytidine or uridine before utilization. Two pathways for metabolism were considered: (i) the reduction of 4-N-hydroxy-cytidine to cytidine followed by deamination, (ii) the direct hydrolysis of hydroxylamine from 4-N-hydroxy-cytidine to yield uridine. A threefold increase in cytidine (deoxycytidine) deaminase (EC 3.5.4.5) activity, when the cells were grown on 4-N-hydroxy-cytidine, suggested the involvement of this enzyme. More direct proof was obtained by purifying the deaminase 185-fold and finding that it released hydroxylamine from 4-N-hydroxy-cytidine at one-fiftieth the rate at which ammonia was removed from cytidine. This result is consistent with the slower rate of growth of the Bu(-) cells on 4-N-hydroxy-cytidine than cytidine and suggests that the second pathway is the major route for utilization of this compound.

Aminohydrolases↗
PubMed1970

Bacteriology of manganese nodules. IV. Induction of an MnO2-reductase system in a marine bacillus.

Bacillus 29, isolated from a ferromanganese nodule from the Atlantic Ocean, was shown to possess an MnO(2)-reductase system which is induced in the presence of manganous ion. Maximal activity of the enzyme system was induced in about 5 hr in the presence of 4.35 mm MnSO(4) and was minimally dependent on the presence of either glucose or peptone and oxygen. Induction of optimal activity required the simultaneous presence of glucose and peptone. At least 30% of maximal activity was induced in 5 hr in the presence of 0.4 mum MnSO(4). Actinomycin D (5 mug/ml) or chloramphenicol (35 mug/ml), when added to the induction medium, inhibited approximately 90% of MnO(2)-reductase synthesis and incorporation of uracil-2-(14)C or leucine-1-(14)C. Cell-free extracts having MnO(2)-reductase activity were prepared by sonic disruption of cell suspensions of induced Bacillus 29. Such extracts used glucose metabolism as a source of electrons. They had an average specific activity of 1.15 nmoles of Mn(II) produced per mg of protein per hr at 25 C. They had a temperature optimum of 18 C for reductase activity and retained 50% of their activity at 4 C, the approximate temperature of the natural habitat of the organism. Extracts were stable for several days at 4 C but rapidly lost over 50% of their activity on freezing and thawing. Over 90% of the activity of the extract could be destroyed by heating in a boiling-water bath for 5 min. At a concentration of 1 mm, HgCl(2) and atebrine dihydrochloride inhibited MnO(2)-reductase activity by at least 50%, but sodium azide was ineffective. The MnO(2)-reductase activity of induced cells and extracts from them was no greater in the absence of oxygen than in its presence, confirming an earlier observation that MnO(2) and O(2) do not compete as terminal electron acceptors in the respiratory activity of this organism.

Bacillus↗
PubMed1968

Bacteriology of manganese nodules: III. Reduction of MnO(2) by two strains of nodule bacteria.

MnO(2) reduction by aerobic growing cultures of Bacillus 29 and coccus 32, isolated from ferromanganese nodules, was assessed for 7 days. A 1-day lag was observed before the onset of MnO(2) reduction by either culture. Addition of HgCl(2) to a final concentration of about 10 M caused a rapid cessation of MnO(2) reduction by the growing cultures. Neither culture reduced MnO(2) when grown under continued anaerobiosis from the start of an experiment. However, if conditions were made anaerobic after MnO(2) reduction was initiated, reduction continued at a rate only slightly lower than that under aerobic conditions. Resting-cell cultures reduced MnO(2) equally well aerobically and anaerobically, provided that ferricyanide was present to serve as electron carrier. These findings showed that oxygen is needed for culture adaptation to MnO(2) reduction, and that oxygen does not interfere with microbial MnO(2) reduction itself. Both cultures caused sharp drops in the pH of the medium during MnO(2) reduction: with coccus 32, during the entire incubation time; with Bacillus 29, for the first 3 days. The E(h) of the medium fluctuated with either culture and never fell below 469 mv with Bacillus 29 and below 394 mv with coccus 32. The rates of glucose consumption and Mn release by Bacillus 29 and coccus 32 were fairly constant, but the rates of lactate and pyruvate production were not. Although acid production undoubtedly helped in the reduction of pyrolusite (MnO(2)) by the bacteria, it did not appear to be important in the reduction of manganese oxide in ferromanganese nodules, as shown by the results with a nodule enrichment.

Journal Article↗