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K S Anderson

Publications and source records attributed to K S Anderson.

64 records · Page 4Linked to original sources
PubMed1990

Observation by 13C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site.

Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by 13C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using [2-13C]PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP (152 ppm) and EPSP bound to the enzyme in a ternary complex with phosphate (161 ppm). These peak assignments account for our quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted; the intermediate could be isolated by quenching with either triethylamine or 0.2 N KOH, allowing direct quantitation of the species bound to the enzyme. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal [Leo et al. (1990) J. Am. Chem. Soc. (in press)]. The rate of formation of the EPSP ketal was very slow, 3.3 X 10(-5) s-1, establishing that it is a side product of the normal enzymatic reaction, probably arising as a breakdown product of the tetrahedral intermediate. A slow formation of pyruvate was also observed and is attributable to the enzymatic hydrolysis of EPSP, with 5% of the enzyme sites occupied by EPSP and hydrolyzing EPSP at a rate of 4.7 X 10(-4) s-1. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, an NMR experiment was performed with an analogue of S3P lacking the 4- and 5-hydroxyl groups.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1988

A tetrahedral intermediate in the EPSP synthase reaction observed by rapid quench kinetics.

Direct evidence for an enzyme-bound intermediate in the EPSP synthase reaction pathway has been obtained by rapid chemical quench-flow studies. The transient-state kinetic analysis has led to the following complete scheme: (formula; see text) Values for all 12 rate constants were obtained. Substrate trapping experiments in the forward and reverse reactions established the kinetically preferred order of binding and release of substrates and products and showed that shikimate 3-phosphate (S3P) and 5-enolpyruvoylshikimate 3-phosphate (EPSP) dissociate at rates greater than turnover in each direction. Pre-steady-state bursts of product formation were observed in the reaction in each direction indicating a rate-limiting step following catalysis. Single turnover experiments with enzyme in excess over substrate demonstrated the formation of a transient intermediate in both the forward and reverse reactions. In these experiments, the enzymatic reaction was observed by employing a radiolabel in the enol moiety of either phosphoenol pyruvate (PEP) or EPSP. The separation and quantitation of reaction products were accomplished by HPLC monitoring radioactivity. The intermediate was observed as the transient production of radiolabeled pyruvate, formed due to the breakdown of the intermediate in the acid quench used to stop the reaction. The intermediate was observed within 5-10 ms after the substrates were mixed with enzyme and decayed in a reaction paralleling the formation of product in each direction. Thus, the kinetics demonstrate directly the kinetic competence of the presumed intermediate. No pyruvate was formed, on a time scale which is relevant to catalysis, after incubation of the enzyme with dideoxy-S3P and PEP or with EPSP in the absence of phosphate; and so, the intermediate does not accumulate under these conditions. The intermediate broke down to form PEP and EPSP in addition to pyruvate when the reaction was quenched with base rather than acid; therefore, the intermediate must contain the elements of each product. Other experiments were designed to measure directly the phosphate binding rate and further constrain the PEP binding rate. The overall solution equilibrium constant in the forward direction was determined to be 180 by quantitation of radiolabeled reactants and products in equilibrium after incubation with a low enzyme concentration. The internal, active site equilibrium constant was obtained by incubation of radiolabeled S3P with excess enzyme and high concentrations of phosphate and PEP to provide the ratio of [EPSP]/[S3P] = 2.3, which is largely a measure of K4.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1988

Tissue-specific induction of murine glutathione transferase mRNAs by butylated hydroxyanisole.

Three murine liver glutathione transferase (GT, EC 2.5.1.18) have been cloned and sequenced. Two of the cDNA clones, pGT875 and pGT55, encode the murine class-mu GT isoenzymes, GT8.7 and GT9.3, respectively. These two cDNA clones share 85% DNA sequence identity with one another, and the GT8.7 sub-unit encoded by pGT875 shares 92% protein sequence identity with the class-mu rat-3 (Yb1) GT subunit. The third cDNA clone, pGT41, encodes a class-alpha GT subunit that shares 96% protein sequence identity with a mouse Ya gene, 95% identity with a rat-1 (Ya) GT subunit, and 70% identity with the rat-2 (Yc) subunit. These cDNA clones and an oligonucleotide derived from the sequence of a rat class-pi cDNA clone were used to measure the induction of the mu, alpha, and pi classes of GT mRNA in different tissues of mice that were fed the dietary antioxidant 2(3)-tert-butyl hydroxyanisole (BHA). These tissues included liver, intestinal mucosa, kidney, lung, spleen, and brain. Class-mu GT mRNAs that hybridize with pGT875 are most abundant in liver and intestinal mucosa but are also found in kidney and lung, and at low levels in brain and spleen. Class-alpha GT mRNAs are most abundant in BHA-induced and uninduced intestinal mucosa, kidney, and induced liver, and were not found in spleen and brain. Class-mu and -alpha GT mRNA levels increased 15- and 50-fold, respectively, in the liver and 15- and 100-fold in intestinal mucosa in response to BHA induction. BHA increases class-mu mRNAs less than 5-fold in the kidney and lung. Class-pi mRNAs were found in all the tissues examined but were much less responsive to BHA induction. The expression of two cytochrome P-450 mRNAs increased 3-5-fold in liver and intestine after BHA induction. Oligonucleotides from divergent portions of the pGT875 and pGT55 cDNA clones have been used to examine the expression of specific mRNAs from individual class-mu GT genes; these experiments suggest that the GT mRNAs expressed in BHA-induced tissues are also expressed in the uninduced tissue. Measurements of transcription rates in isolated nuclei showed that increased GT mRNA levels are due to increased rates of transcription.

Amino Acid Sequence↗
PubMed1988

Evaluation of 5-enolpyruvoylshikimate-3-phosphate synthase substrate and inhibitor binding by stopped-flow and equilibrium fluorescence measurements.

The binding of substrates and the herbicide N-(phosphonomethyl)glycine (glyphosate) to enolpyruvoylshikimate-3-phosphate (EPSP) synthase was evaluated by stopped-flow and equilibrium fluorescence measurements. Changes in protein fluorescence were observed upon the binding of EPSP and upon the formation of the enzyme-shikimate 3-phosphate-glyphosate ternary complex; no change was seen with either shikimate 3-phosphate (S3P) or glyphosate alone. By fluorescence titrations, the dissociation constants were determined for the formation of the enzyme binary complexes with S3P (Kd,S = 7 +/- 1.2 microM) and EPSP (Kd,EPSP = 1 +/- 0.01 microM). The dissociation constant for S3P was determined by competition with EPSP or by measurements in the presence of a low glyphosate concentration. At saturating concentrations of S3P, glyphosate bound to the enzyme--S3P binary complex with a dissociation constant of 0.16 +/- 0.02 microM. Glyphosate did not bind significantly to free enzyme, so the binding is ordered with S3P binding first: (formula; see text) where S refers to S3P, G refers to glyphosate, and E.S.G. represents the complex with altered fluorescence. The kinetics of binding were measured by stopped-flow fluorescence methods. The rate of glyphosate binding to the enzyme--S3P complex was k2 = (7.8 +/- 0.2) X 10(5) M-1 s-1, from which we calculated the dissociation rate k-2 = 0.12 +/- 0.02 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1988

Duration of Alphavirus neutralizing antibody in naturally infected birds.

Native birds, mostly passerine species, ecologically associated with Culiseta melanura, the enzootic vector of eastern equine encephalomyelitis and Highlands J viruses in the eastern United States, were examined over a 12-year period in southeastern Massachusetts. These studies concentrated on those individual birds known, by banding returns, to be residents of large wooded swamps where both eastern equine encephalomyelitis and Highlands J viruses were known to be enzootic. Of 8,417 birds sampled, 1,227 (14.6%) were recaptured one or more times (mean 2.7 times). Antibody profiles on individuals nesting or feeding in enzootic areas were determined from serial blood samples drawn from these recaptured birds. The duration of detectable neutralizing antibody in these birds was found to be ephemeral in some species (e.g., black-capped chickadees) and extremely longlasting in others (e.g., gray catbirds, swamp sparrows). The significance of these findings to arbovirus surveillance programs is discussed.

Alphavirus↗
PubMed1985

Antagonism of prostaglandin-mediated responses in platelets and vascular smooth muscle by 13-azaprostanoic acid analogs. Evidence for selective blockade of thromboxane A2 responses.

Studies were undertaken to examine the pharmacological properties and stereochemical requirements of a limited series of prostanoic acid analogs for inhibition of arachidonic acid (AA) and/or endoperoxide (U46619)-mediated responses in human platelets and rat aorta. To assess the role of stereochemistry, a set of trans- and cis-isomers of 13-azaprostanoic acid (APA) and 11a-homo-13-azaprostanoic acid (HAPA) were prepared. Each prostanoic acid analog blocked AA- or U46619-induced aggregatory and secretory responses in platelets, and U46619-mediated contractions of rat aorta in a concentration-dependent manner (0.1 to 100 microM). The azaprostanoic acid analogs blocked responses to both inducers of platelet activation with IC50 values ranging from 3.4 to 27.5 microM. Trans-APA was about 2- to 3-fold more active as an antagonist of serotonin release induced by AA or U46619 than the remaining analogs. The rank order of inhibitory potency (IC50; microM) for these analogs against U46619-induced serotonin release in human platelets was trans-APA (3.4) greater than cis-APA (8.9) = cis-HAPA (8.7) = trans-HAPA (9.1). Concentrations of the prostanoic acid analogs required to block these responses to AA and U46619 were similar, and the highest concentration used (100 microM) did not modify AA-induced malondialdehyde production in human platelet preparations. In contrast, the isomers of APA and HAPA were equally active as antagonists of U46619-induced contractions of rat vascular tissue, possessing KB values varying from 7.1 to 13.2 microM. Each azaprostanoic acid analog shifted the concentration-response curve of U46619 in rat aorta to the right, indicating a competitive-type inhibition. In addition, the azoprostanoic acid analog (U51605) was a more potent competitive antagonist of U46619 in this preparation and possessed an average pKB value of 6.18. In summary, the results show that (1) expansion of the five-membered ring of APA to the six-membered ring analogs (HAPA) led to a retention of potent inhibitory activity against U46619 in human platelets and rat vascular smooth muscle, (2) the antiaggregatory and antisecretory actions of the azaprostanoic acid analogs were mediated by a blockade of the responses to AA and U46619, and not by an inhibition of AA metabolism, (3) the blocking activity for the APA isomers was stereoselective (trans greater than cis) whereas the isomers of HAPA were equally effective as inhibitors of platelet function; and (4) these azaprostanoic acid analogs act as selective endoperoxide (U46619)/thromboxane A2 antagonists in these two tissues.

Adult↗
PubMed1982

Conformational requirements of alpha 2-adrenergic receptors.

The conformationally restrained trans-extended and cis-folded isomers of 2-(3,4-dihydroxyphenyl)cyclobutylamine were used to establish the conformational requirements of presynaptic alpha 2-adrenergic receptors in the field-stimulated guinea pig ileum. The trans-extended isomer produced a concentration-dependent inhibition of the twitch response with an EC50 of 34 microM. The cis-folded analogue failed to produce a significant inhibition of the twitch response at concentrations up to 1 mM. These results suggest that the presynaptic alpha 2-adrenergic receptor prefers phenethylamines in the trans-extended conformation over the cis-folded conformation. The conformational requirements of alpha 1- and alpha 2-adrenergic receptors appear to be similar.

Animals↗
PubMedDate not supplied

Mechanistic studies to understand the inhibition of wild type and mutant HIV-1 reverse transcriptase by Carbovir-triphosphate.

Abacavir (1592U89) has recently been approved by the FDA for treatment of HIV infection. Transient kinetic studies were carried out to better understand the interaction of the active metabolite of Abacavir (Carbovir-triphosphate) with wild type and mutant HIV-1 reverse transcriptase. Some of the data is summarized and used as a basis for discussion of inhibition by CBVTP and previously studied nucleoside analogs.

Anti-HIV Agents↗