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Biomedical subjects

D B Northrop

Publications and source records attributed to D B Northrop.

At least 55 records · Page 3Linked to original sources
PubMed1983

Purification of reduced nicotinamide adenine dinucleotide by ion-exchange and high-performance liquid chromatography.

Combining ion-exchange (AG MP-1) and reversed-phase (C-18) partition chromatography accomplishes a higher degree of purification of NADH than either method can provide alone. Final elution in 95% ethanol, dehydration with anhydrous sodium sulfate, and storage in dry 1,2-propanediol over molecular sieves prevents hydrolysis of the purified dinucleotide.

Chromatography, High Pressure Liquid↗
PubMed1981

Minimal kinetic mechanism and general equation for deuterium isotope effects on enzymic reactions: uncertainty in detecting a rate-limiting step.

A general equation is proposed for representing the kinetic functions which govern the expression of an isotope effect on the maximal velocity of an enzyme-catalyzed reaction. The origin and form of the functions are illustrated by examining a series of enzymatic mechanisms of progressively increasing complexity. The number of functions similarly increase, reaching a limit of three, with differing thermodynamic and kinetic properties. Further expansion of mechanisms causes an orderly and predictable algebraic expansion of each function, making it possible to write out, by simple inspection, the kinetic equation describing an isotope effect expressed on the maximal velocity for any enzymatic mechanism in which the isotope perturbs a single reactive step. The functions are interactive and allow for the possibility that an isotope effect on Vmax may be independent of the rate of a second, isotopically insensitive step, be it infinitely fast or slow. This allowance leads to an uncertainty of the ability of an isotope effect to detect a rate-limiting step, and the unequal distribution of kinetic and thermodynamic properties among three functions leads to an inadequacy of the singular concept of a rate-limiting step to serve as a basis for interpreting isotope effects on enzyme-catalyzed reactions. A minimal mechanism for consideration of isotope effects is proposed in order to embrace all three functions. It consists of a single catalytic step which is isotopically sensitive and reversible, two reversible precatalytic steps, and one reversible postcatalytic step, plus steps for binding and release of substrates and products.

Deuterium↗
PubMed1980

Kinetic isotope effects in cytochrome P-450-catalyzed oxidation reactions. Intermolecular and intramolecular deuterium isotope effects during the N-demethylation of N,N-dimethylphentermine.

Two N,N-dimethylphentermine (N,N-dimethyl-2-amino-2-methyl-3-phenylpropane) substrates differing in deuterium substitution have been used to determine the intermolecular and intramolecular isotope effects associated with the cytochrome P-450-dependent N-demethylation of this substrate. No intermolecular isotope effect was observed in Vmax or Vmax/Km when the reaction rates for this substrate were compared to those for the substrate in which both N-methyl groups contained deuterium. In contrast, identical isotope effects of 1.6 to 2.0 were observed in both Vmax and Vmax/Km when this reaction was studied with a substrate in which only one of the two N-methyl groups was substituted with deuterium. Furthermore, both the intermolecular and intramolecular isotope effects were independent of the cytochrome P-450/NADPH-cytochrome P-450 reductase mole ratio. From these data, it is concluded that: 1) the carbon-hydrogen bond cleavage step does not contribute significantly to Vmax; 2) the contribution of the carbon-hydrogen bond cleavage step to Vmax is not detectably increased through changes in the cytochrome P-450/NADPH-cytochrome P-450 reductase mole ratio; 3) the N-methyl groups are free to exchange at the enzyme active site. The basis for these conclusions is the proposal of a new kinetic model for interpretation of intramolecular isotope effects which shows that intramolecular isotope effects are not necessarily equal to intrinsic isotope effects and, in fact, may be much smaller.

Animals↗
PubMed1979

Spectrophotometric assay for amikacin using purified kanamycin acetyltransferase.

A rapid spectrophotometric assay has been developed for measuring the concentrations of amikacin and related antibiotics in serum. The assay uses a purified enzyme from R-factor E. coli which acetylates amikacin with the production of coenzyme A, the latter in turn being reacted with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore, that is measured in the visible spectrum. The system complements an earlier assay for gentamicin-related antibiotics thereby facilitating the rapid measurement of the concentrations of all clinically important aminoglycosides in serum.

Acetyltransferases↗
PubMed1979

Synthesis of a tight-binding, multisubstrate analog inhibitor of gentamicin acetyltransferase I.

Gentamicin acetyltransferase I will catalyze acyl transfer from chloroacetylcoenzyme A to form 3-N-chloroacetylgentamicin. This product can be linked to coenzyme A to form a multisubstrate analog by nucleophilic displacement of the chlorine by the sulfur of coenzyme A. The analog can be purified by selective binding to cationic and anionic ion exchange resins. Kinetic analysis of a time-dependent onset and reversal of inhibition of gentamicin acetyltransferase I by the purified multisubstrate analog yields an inhibition constant of 5 approximately 20 x 10(-10) M. The inhibitor does not potentiate antibiotic activity against resistant Escherichia coli. Nevertheless, the effectiveness of the tight-binding between the enzyme and the multisubstrate analog demonstrates that inhibitors of resistance can be designed and prepared by specific enzymatic synthesis.

Acetyltransferases↗
PubMed1978

Kinetic mechanisms of gentamicin acetyltransferase I. Antibiotic-dependent shift from rapid to nonrapid equilibrium random mechanisms.

Initial velocity, product, dead-end, and substrate inhibition studies are described for gentamicin acetyltransferase I in the forward reaction. Initial velocity patterns were linear with tobramycin (Km acetyl-CoA = 1.7 micron, Km tobramycin = 1.6 micron), concave upward with sisomicin (V/K.Et = 1.7 X 10(7) M-1 s-1, indicative of nonrapid equilibrium conditions), and showed partial uncompetitive substrate inhibition with gentamicin C1a (Km acetyl-CoA = 1.3 micron, Km gentamicin C1a = 0.12 micron, KI = 6.5 micron). Product inhibition by CoA and acetyltobramycin consists of two competitive and two noncompetitive patterns. Dead-end inhibition by butyryl-CoA and neomycin consists of two competitive and two uncompetitive patterns. However, the uncompetitive pattern between neomycin and acetyl-CoA became noncompetitive when sisomicin rather than tobramycin was the nonvaried substrate. These results are consistent with a Random BiBi mechanism with synergism between binding sites which is nonrapid equilibrium with gentamicin C1a and sisomicin and rapid equilibrium with poor substrates such as tobramycin. The substrate inhibition by gentamicin C1a arises from a reduction in the rate of product (CoA) release which is partially rate-determining under nonrapid equilibrium conditions.

Acetyltransferases↗
PubMed1976

Purification and properties of gentamicin acetyltransferase I.

Gentamicin acetyltransferase I is induced 13-fold in R factor resistant Escherichia coli by high concentrations (1 mg/ml) of gentamicin in the growth medium. The enzyme is maximally released from bacteria by osmotic shock in late-log phase, unlike previously studied periplasmic enzymes. Streptomycin sulfate and ammonium sulfate precipitations of shockate followed by affinity and ion-exchange chromatography recover 51% of the induced enzyme with a 360-fold increase in purity (12% of 4400-fold, uninduced). The purified enzyme appears homogeneous by six criteria, the first aminoglycoside inactivating enzyme so purified. Sodium dodecyl sulfate electrophoresis, amino acid analysis, and sedimentation analyses indicate a tetrameric protein of 63000 molecular weight. The protein does not contain tryptophan. Kinetic analyses yield apparent values of: Vmax = 3.4 +/- 0.2 mumol per min mg at pH 8 (optimum), Km (acetyl-CoA) = 3.9 +/- 0.2 muM, Km (gentamicin Cla) = 0.3 +/- 0.08 muM, KI (gentamicin substrate inhibition) = 160 +/- 29 muM. The activity of the enzyme is stable to a variety of conditions, including lyophilization and prolonged storage, and can be monitored by two convenient spectrophotometric assays.

Acetyltransferases↗
PubMed1975

A spectrophotometric assay for gentamicin.

A rapid and accurate spectrophotometric assay has been developed for the determination of blood werum levels of gentamicin and related antibiotics. The assay uses a purified enzyme from Escherichia coli JR88/C600 that acetylates gentamicin with the production of coenzyme A, linked to a chemical reaction with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore, monitored in the visible spectrum. The system provides advantages of speed, cost, convenience, accuracy, and enzyme stability to the desirable characteristics encountered with previous enzymatic methods.

Acetyltransferases↗