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Biomedical subjects

C T Walsh

Publications and source records attributed to C T Walsh.

At least 325 records · Page 18Linked to original sources
PubMed1981

Reconstitution of Escherichia coli membrane vesicles with D-amino acid dehydrogenase.

When purified D-amino acid dehydrogenase [Olsiewski, P. J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 255, 4487] is incubated with right-side-out membrane vesicles from Escherichia coli, the enzyme binds to the membrane in a time- and concentration-dependent manner. As a result, the vesicles acquire the ability to oxidize D-alanine and catalyze D-alanine-dependent active transport. Similarly, incubation of D-amino acid dehydrogenase with inside-out vesicles results in binding of enzyme and D-alanine oxidase activity. Antibody inhibition studies indicate that the enzyme is bound exclusively to the inner cytoplasmic surface of the membrane in native vesicles (i.e., membrane vesicles prepared from cells induced for D-amino acid dehydrogenase). In contrast, similar studies with reconstituted vesicles demonstrate that enzyme binds to the surface exposed to the medium regardless of the orientation of the membrane. Thus, enzyme bound to right-side-out vesicles is located on the opposite side of the membrane from where it is normally found. Remarkably, in the presence of D-alanine, reconstituted right-side-out and inside-out vesicles generate electrochemical proton gradients of similar magnitude but opposite polarity, indicating that enzyme bound to either surface of the membrane is physiologically functional. The results suggest that vectorial proton translocation via the respiratory chain occurs at a point distal to the site where electrons enter the respiratory chain from the primary dehydrogenase, a conclusion that is inconsistent with the notion that the dehydrogenase forms part of a proton-translocating loop.

Alanine↗
PubMed1980

Increased activation of 1-beta-D-arabinofuranosylcytosine by hydroxyurea in L1210 cells.

Hydroxyurea (0.1 to 10 mM), incubated with L1210 cells in the presence of 1 microM 1-beta-D-arabinofuranosylcytosine (ara-C), produced a concentration- and time-dependent increase in levels of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP). This effect was abolished upon removal of hydroxyurea from cells. The enhancement of ara-CTP by hydroxyurea was observed for concentrations of ara-C ranging from 0.1 to 100 microM. These changes were not associated with alteration in the activity of deoxycytidine kinase, measured in extracts from L1210 cells both with and without deoxycytidine triphosphate. Hydroxyurea did, however, increase the apparent maximum velocity of this enzyme when determined with intact cell preparations, a finding consistent with alteration in endogenous regulation of this enzyme. Four-hr i.v. infusion of hydroxyurea (192 mg/kg/hr) with ara-C (1 mg/kg/hr) into ascites tumor-bearing mice also resulted in increased L1210 cell concentrations of ara-CTP. ara-C levels in plasma and L1210 cells were unaffected by hydroxyurea. Therefore, the enhancement of ara-CTP concentration in vivo, like in vitro, resulted from changes in ara-C conversion in the L1210 cells.

Animals↗
PubMed1979

Methods for the analysis of intestinal function.

The intestinal tract, an organ of considerable complexity, requires application of numerous techniques for analysis of its physiology and perturbations by toxicologic agents. This review describes the methodology of importance in analysis of the absorptive function of the intestine and the transit of intestinal contents. Methods for studying absorption are categorized according to the technique for administering the test substance such as inclusion in the diet or by gastric and intestinal placement and the method of quantitating the degree of absorption such as determining the appearance of a test substance in systemic fluids or its disappearance from its site of administration in the intestine. In vitro techniques which have no in vivo analogs, such as the use of the everted sac, are briefly described and their limitations emphasized. Procedures of importance in the clinical diagnosis of malabsorption or in the experimental analysis of absorptive function in man are included and distinguished from techniques used in animal models. In addition, methods for studying aspects of gastrointestinal motility, including the use of luminal markers and analysis of the contractile and electrical activity of intestinal smooth muscle, are reviewed.

Animals↗
PubMed1977

Inactivation of bacterial D-amino acid transaminases by the olefinic amino acid D-vinylglycine.

D-Vinylglycine (2-amino-3-butenoate) functions as a transamination substrate and irreversible inactivator of the homogeneous pyridoxal phosphate-dependent D-amino acid transaminases from Bacillus subtilis and Bacillus sphaericus. In the absence of alpha-ketoglutarate as co-substrate, vinyl-glycine causes little if any inactivation of either enzyme; in the presence of excess alpha-ketoglutarate, both enzymes are inactivated with pseudo-first order kinetics. The limiting rate constant for inactivation of the B. sphaericus enzyme is 1.9 min-1, for the B. subilis enzyme it is 0.36 min-1. The number of catalytic events before inactivation is about 450 for the B. sphaericus enzyme and about 800 for the B. subtilis enzyme; that is, about 0.2% inactivation in each catalytic cycle for the former enzyme and 0.15% for the latter. Comparisons are made with the L-aspartate amino-transferase from pig heart which is inactivated completely in one catalytic cycle and the L-alanine aminotransferase which is not inactivated in many cycles. Comparisons are also made between the likely mode of D-transaminase inactivation produced by vinylglycine and the mode of inactivation induced by beta-chloro-D-alanine.

Amino Acids↗
PubMed1976

Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate] 2-hydroxy-3-butynoate.

2-Hydroxy-3-butynoate is both a substrate and an irreversible inactivator of the flavoenzyme L-lactate oxidase. The partitioning between catalytic oxidation of 2-hydroxy-3-butynoate and inactivation of the enzyme is determined by the concentration of the second substrate, O2. Rapid reaction studies show the formation of an intermediate which is common to both the oxidation and inactivation pathways. This intermediate appears to be a charge-transfer complex between enzyme-reduced flavin and 2-keto-3-butynoate. It is characterized by a long-wavelength absorbing band (gamma(max) 600 nm) and lack of fluorescence, making it easily distinguished from the subsequently formed inactivated enzyme, which has no long wavelength absorption (gamma(max) 318, 368 nm) and which is strongly fluorescent. Inactivation is also accomplished by reaction of the reduced enzyme with 2-keto-3-butynoate. The absorbance and fluorescence characteristics of the inactivated enzyme are similar to those of a model compound, C(4a), N(5)-propano-bridged FMN bound to apolactate oxidase. That the modified chromophore of the inactivated enzyme is an adduct involving both the C(4a) and N5 positions is further supported by the spectral and fluorescence changes resulting from treatment of the inactivated enzyme with borohydride.

Anaerobiosis↗
PubMed1976

The structure of the covalent flavin adduct formed between lactate oxidase and the suicide substrate 2-hydroxy-3-butynoate.

2-Hydroxy-3-butynoic acid is a suicide substrate for Mycobacterium smegmatis lactate oxidase. Inactivation occurs by covalent modification of enzyme-bound FMN and does not involve labeling of the apoprotein. The spectrum of the enzyme bound adduct suggests that it is a 4a, 5-dihydroflavin derivative. When this adduct is released from the enzyme, a complex mixture of unstable compounds is obtained. When the initially formed enzyme-bound adduct is reduced with NaBH4, a major stable species can be resolved from the enzyme and can be isolated and purified. The structure was established by appropriate isotope substitutions. Fourier transform NMR spectroscopy, chemical reactivity, and synthesis of a model compound. The structure of the isolated adduct is structure II, Scheme II. The structure proposed for the adduct initially formed on the enzyme is structure VII, Scheme II.

Binding Sites↗
PubMed1976

Escherichia coli glyoxalate carboligase. Properties and reconstitution with 5-deazaFAD and 1,5-dihydrodeazaFADH2.

Glyoxalate carboligase (EC 4.1.1.47) has been purified to electrophoretic homogeneity from Escherichia coli. The enzyme was found to be a dimer of subunits of identical molecular weight of 68,000. Resolution of the holoenzyme into apoenzyme and FAD led to a dissociation of the dimer into monomers. The apoenzyme could be reconsitituted to full catalytic activity with FAD or the flavin coenzyme analogue 5-deazaFAD. Reconstitution of the apoenzyme with the reduced flavin analogue 1,5-dihydro-5-deazaFADH2 led to the recovery of 50% of enzymatic activity. The reconstitution of apoglyoxalate carboligase with all three coenzymes followed Michaelis-Menten kinetics with Km values of 0.25, 0.74, and 0.72 muM for FAD deazaFAD, and deazaFADH2, respectively.

Binding Sites↗
PubMed1975

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.

Amino Acid Oxidoreductases↗
PubMed1975

Studies of the enterohepatic circulation of morphine in the rat.

The disposition of 14C-morphine was studied after gastrointestinal and subcutaneous administration to female Sprague-Dawley rats. Morphine was found to be rapidly and nearly completely absorbed from the small intestine. After s.c. administration, approximately half the 5 mg/kg dose of morphine was excreted via the bile into the intestinal tract, largely as morphine glucuronide. Unlike morphine, the glucuronide conjugate in bile was poorly absorbed from the small intestine where its hydrolysis occurred slowly. After administration into the cecum, however, hydrolysis of the conjugate was rapid, and the rate of absorption of radioactive material was similar to that of free morphine. Treatment of rats with lincomycin (500 mg/1 in drinking water and 25 mg twice a day by gastric intubation for 4 days) significantly decreased cecal hydrolysis of the conjugate and the cecal absorption of radioactive material. Lincomycin treatment also increased fecal excretion of the conjugate in rats given morphine s.c. (5 mg/kg). These findings indicated that hydrolysis of morphine glucuronide is dependent upon the status of enteric bacteria and is a prerequisite to the enterohepatic circulation of morphine. In addition, after lincomycin treatment, a greater percentage of radioactivity excreted in the urine was associated with free morphine. This finding could be expained by the demonstration of pH dependence of renal excretion of morphine and a first-pass effect for this drug.

Ammonium Chloride↗
PubMed1974

Determination of the absolute number of Escherichia coli membrane vesicles that catalyze active transport.

Transport of vinylglycolate (2-hydroxy-3-butenoic acid) via the lactate transport system is the limiting step for covalent labeling of membrane vesicles prepared from E. coli ML 308-225. Thus, the rate and extent of vinylglycolate labeling is stimulated about 10-fold by ascorbate-phenazine methosulfate, and stimulation is abolished by 2,4-dinitrophenol and by phospholipase treatment, neither of which affect the rate of vinylglycolate oxidation. [(3)H]Vinylglycolate of high specific activity has been prepared, and vesicles have been labeled with this compound in the presence of ascorbate-phenazine methosulfate. Examination of these preparations by high resolution radioautography in the electron microscope demonstrates that virtually all of the vesicles are labeled. The experiments provide a strong indication that most, if not all, of the membrane vesicles in these preparations catalyze active transport.

Ascorbic Acid↗