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Biomedical subjects

F B Rudolph

Publications and source records attributed to F B Rudolph.

At least 55 records · Page 3Linked to original sources
PubMed1989

Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicum (ATCC 824).

Two butanol dehydrogenases with different cofactor requirements and different pH ranges have been detected in Clostridium acetobutylicum ATCC 824. The NADH-dependent butanol dehydrogenase (NADH-BDH) was purified to near homogeneity and characterized. One striking feature of the enzyme is that Zn2+ was needed to obtain a significant recovery during purification. The enzyme was a dimer composed of two subunits with subunit molecular mass of 42 kDa and a native molecular mass of 82 +/- 2 kDa. The kinetics were studied in the direction of the reduction of butyraldehyde. Inhibition studies with S-NADH and butanol indicate that the NADH-BDH follows an ordered bibi mechanism with kinetic constants of 4.86 s-1, 0.18 mM, and 16 mM for Kcat, KNADH, and Kbutyraldehyde, respectively. Activity in the reverse direction was 50-fold lower than that in the forward direction. The NADH-BDH had higher activity with longer chained aldehydes and was inhibited by metabolites containing an adenine moiety.

Alcohol Oxidoreductases↗
PubMed1989

Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis.

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.

Acetyltransferases↗
PubMed1989

Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids.

Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30 degrees C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 microM, depending on the acid substrate. The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.

Biological Transport↗
PubMed1989

Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes.

Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.

Journal Article↗
PubMed1988

Preliminary X-ray analysis of crystals of murine adenosine deaminase.

We have obtained single crystals of a cloned mammalian adenosine deaminase (Mr = 41,000), a key enzyme in purine degradation and in normal development of the immune system, that are suitable for high-resolution structural analysis. The crystals belong to the space group C2 with unit cell parameters a = 101.68 A (1 A = 0.1 nm), b = 94.38 A, c = 85.51 A, and beta = 96.54 degrees. The asymmetric unit contains two enzyme molecules.

Adenosine Deaminase↗
PubMed1988

Thiolase from Clostridium acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents.

Thiolase (acetyl-coenzyme A [CoA] acetyltransferase, E.C. 2.3.1.19) from Clostridium acetobutylicum ATCC 824 has been purified 70-fold to homogeneity. Unlike the thiolase in Clostridium pasteurianum, this thiolase has high relative activity throughout the physiological range of internal pH of 5.5 to 7.0, indicating that change in internal pH during acid production is not an important factor in the regulation of this thiolase. In the condensation direction, the thiolase is inhibited by micromolar levels of CoA, and this may be an important factor in modulating the net condensation of acetyl-CoA to acetoacetyl-CoA. Other cofactors and metabolites that were tested and shown to be inhibitors are ATP and butyryl-CoA. The native enzyme consists of four 44,000-molecular-weight subunits. The kinetic binding mechanism is ping-pong. The K(m) value for acetyl-CoA is 0.27 mM at 30 degrees C and pH 7.4. The K(m) values for sulfhydryl-CoA and acetoacetyl-CoA are, respectively, 0.0048 and 0.032 mM at 30 degrees C and pH 8.0. The active site apparently contains a sulfhydryl group, but unlike other thiolases, this thiolase is relatively stable in the presence of 5,5'-dithiobis(2-nitrobenzoic acid). Studies of thiolase specific activity under various types of continuous fermentations show that regulation of this enzyme at both the genetic and enzyme levels is important.

Journal Article↗
PubMed1986

Isotope exchange at equilibrium studies with rat muscle adenylosuccinate synthetase.

The kinetic mechanism of rat muscle adenylosuccinate synthetase was studied by determining the rates of isotope exchange at equilibrium. A random sequential binding mechanism was indicated for both the forward and reverse reactions. Aspartate, adenylosuccinate, GDP, and Pi were determined to bind in rapid equilibrium. GTP exchanges with both GDP and Pi at the same rate, which is similar to the exchange rate of IMP with adenylosuccinate. Aspartate exchanges with adenylosuccinate at a higher rate than does IMP over the range of concentrations tested. The slower IMP and GTP exchange rates suggest a forward binding mechanism containing a preferred path in which the quaternary complex is most often formed by aspartate binding to the E-GTP-IMP complex. This preferred path is consistent with an interaction between IMP and GTP in the absence of aspartate as determined by isotope scrambling experiments [Bass, M. B., Fromm, H. J., & Rudolph, F. B. (1984) J. Biol. Chem. 259, 12330-12333]. However, the products of such an interaction are tightly bound to the enzyme as no partial exchange reactions between adenylosuccinate and aspartate in the presence or absence of Pi were detected.

Adenylosuccinate Synthase↗
PubMed1986

Influence of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice.

Although enzyme defects in purine metabolism have revealed the importance of these substrates to maintenance of a normal immune response, the role of exogenous nucleotides on the cells that mediate the host defense system has remained largely unexplored. Recent investigations have revealed that dietary nucleotides are vital to the maintenance of cell-mediated responses to antigen stimulation. To test the influence of dietary nucleotide deprivation on resistance to infection, Balb/c mice were maintained on chow, a nucleotide-free (NF) diet, or an NF diet repleted with adenine, uracil, or RNA. Mice on the NF diet suffered 100% mortality following intravenous challenge with Staphylococcus aureus, while chow-fed and RNA- or uracil-repleted mice demonstrated significantly greater resistance to this bacterial challenge. Macrophages from mice on the NF diet had decreased phagocytic activity as measured by uptake of radiolabeled bacteria compared with mice maintained on the NF diet supplemented with adenine, uracil, or RNA. No change in S aureus antibody response was noted on the various diets. Although the mechanism of this suppression of nonspecific immunity remains unclear, provision of nucleotides to defined diets appears vital to maintain host resistance to bacterial challenge.

Agglutination Tests↗
PubMed1986

Role of culture conditions and exposure duration in determining sensitivity of human bone marrow progenitor cells to methotrexate.

The effect of drug concentration, exposure duration, and culture conditions on the cytotoxic activity of methotrexate (MTX) on normal granulocyte-macrophage colony-forming units in culture (GM-CFUC) was studied using a bilayer soft agar system with nucleoside-free medium. The degree of inhibition of colony formation depended on the type of serum supplementation. A 1 h or 2 h pulse treatment with 2 X 10(-4) M (100 micrograms/ml) MTX failed to kill GM-CFUC, when the cells were subsequently plated in a system containing 15% undialyzed fetal bovine serum (FBS). For continuous exposure the observed LD50 of MTX in the agar system was higher than 10(-4) M for 15% undialyzed FBS, 10(-5) M for 15% dialyzed FBS plus 0.25% undialyzed FBS, 10(-6) M for 15% dialyzed FBS, and 10(-8) M for 15% undialyzed horse serum. The difference for dialyzed FBS versus horse serum can be explained by differences in nucleoside concentrations. The difference for dialyzed FBS versus horse serum may be secondary to an enhancer of MTX in horse serum. For studying MTX sensitivity of human tumor cells in vitro, we suggest testing conditions that lie within the dose survival curve of GM-CFUC.

Culture Media↗
PubMed1985

Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line.

Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.

Adenosine↗
PubMed1985

Inhibition of fumarase by S-2,3-dicarboxyaziridine.

S-2,3-Dicarboxyaziridine was found to be a potent competitive inhibitor (Ki = 0.08 microM) of fumarase from pig heart. The aziridine did not inactivate the enzyme or exhibit any observable substrate activity. It is likely that it functions as a transition state analogue mimicking the carbanion intermediate found in the normal catalytic reaction. The aziridine inhibited fumarate utilization in ruptured but not intact mitochondria.

Aziridines↗
PubMed1985

Dietary nucleotides, a requirement for helper/inducer T lymphocytes.

Previous investigations have revealed that dietary nucleotide restriction delays the onset of primary murine cardiac allograft rejection and acute graft-versus-host disease followed H-2-incompatible bone marrow transplantation, suppresses sensitization to intradermally injected antigens and suppresses in vivo and in vitro lymphocyte proliferation to alloantigen or lectin stimulation. To determine the mechanisms responsible for these phenomena, BALB/c mice were placed on chow (F), nucleotide free (NF) diet, or NF diet supplemented with 0.25% RNA (NFR), with 0.6% adenine (NFA), or with 0.06% uracil (NFU). Following four weeks of dietary equilibrium, splenic lymphocytes harvested from naive or immunostimulated mice in the various dietary groups were stained with monoclonal antibodies directed Lyt 1, Lyt 2, 3, or surface mouse immunoglobulin (IgG) surface markers. While naive animals demonstrated no differences in lymphocyte subpopulations between groups, following complete Freund's adjuvant (CFA) stimulation, splenic lymphocytes for NF mice demonstrated 27.3 +/- 1.7% Lyt 1+ cells compared with F (32.6 +/- .04%) and NFR mice (33.2 +/- 1.2%) (P less than 0.02). Restriction of dietary nucleotides affected not only phenotypes of T lymphocytes, but also T cell function. Following conconavalin A stimulation of irradiated splenic lymphocytes, IL-2 production was decreased in NF mice compared with the F control group (P less than 0.01). The RNA-repleted diet maintained normal IL-2 production, while addition of adenine or uracil alone did not. Finally, NF diets adversely affected host resistance to the opportunistic pathogen Candida albicans. Following inoculation with 0.25 X 10(6) organisms NF or NFA-fed hosts succumbed more rapidly than F, NFR, or NFU fed hosts (P less than 0.001). These data suggest that helper/inducer T lymphocytes require exogenous nucleotides to respond normally following immune stimulation. Uracil may be the critical substrate, based upon the studies of Candida resistance. By understanding the metabolic basis of NFD-induced immunosuppression, the role of dietary nucleotides in combatting infection and alloantigen rejection can be more clearly defined.

Adenine↗
PubMed1984

The mechanism of the adenylosuccinate synthetase reaction as studied by positional isotope exchange.

In an attempt to gain insight into the mechanism of the rat muscle adenylosuccinate synthetase reaction, experiments using the technique of positional isotope exchange (isotope scrambling) were undertaken. [gamma-18O]GTP was prepared and incubated with Mg2+ and the synthetase in the presence of various ligands. Positional isotope exchange occurred, as measured by nuclear magnetic resonance spectroscopy, when IMP was present. In the absence of IMP, with or without aspartate or succinate, the [gamma-18O]GTP did not exhibit scrambling. These results suggest that the adenylosuccinate synthetase reaction involves the participation of 6-phosphoryl-IMP as an obligatory intermediate. On the basis of experiments carried out in our laboratory as well as in others, we believe the GDP remains bound to the enzyme until the product, adenylosuccinate, is formed. All products may then dissociate randomly from the enzyme. The positional isotope exchange experiments, along with initial-rate experiments carried out in our laboratory, serve to explain the lack of partial exchange reactions associated with the synthetase (Fromm, H. J. (1958) Biochim. Biophys. Acta 29, 255-262), as well as the net inversion of configuration when chiral thio-GTP is converted to thiophosphate (Webb, M. R., Reed, G. H., Cooper, B. F., and Rudolph, F. B. (1984) J. Biol. Chem. 259, 3044-3046).

Adenylosuccinate Synthase↗
PubMed1984

The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion.

The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate.

Adenylosuccinate Synthase↗