Edwin T. Harper

Effects of ethanol on cocaine metabolism: Formation of cocaethylene and norcocaethylene

The coabuse of cocaine and ethanol occurs with high frequency and increases the risk of cocaine-related morbidity and mortality. The mechanisms mediating the toxic interactions of cocaine and ethanol are not clearly defined. This study examined the effects of acute ethanol administration on the metabolism of cocaine in the male Wistar rat. Intraperitoneal administration of 2 g/kg ethanol 30 min prior to administration of 25 mg/kg cocaine resulted in the formation of two ethylated derivatives of cocaine, benzoylecgonine ethyl ester (cocaethylene) and benzoylnorecgonine ethyl ester (norcocaethylene) in liver, brain, and serum. Fifteen minutes after cocaine administration, the tissue levels of cocaethylene were 22, 10, and 9% of the cocaine recovered from liver, serum, and brain, respectively. Ethanol pretreatment increased cocaine concentrations in liver and benzoylnorecgonine concentrations in liver and serum. The increased morbidity and hepatotoxicity seen with acute combined administration of cocaine and ethanol may be due to the formation of the toxic ethylated and N-demethylated metabolites of cocaine. Ethanol pretreatment decreased benzoylecgonine concentrations in serum and liver. The most important consequence of ethanol-induced inhibition of the normally rapid hydrolysis of cocaine to benzoylecgonine may be a decrease in benzoylecgonine-mediated vasoconstriction.

Structural and mechanistic similarities of 6-phosphogluconate and 3-hydroxyisobutyrate dehydrogenases reveal a new enzyme family, the 3-hydroxyacid dehydrogenases

Rat 3‐hydroxyisobutyrate dehydrogenase exhibits significant amino acid sequence homology with 6‐phosphogluconate dehydrogenase, d‐phenylserine dehydrogenase from Pseudomonas syringae, and a number of hypothetical proteins encoded by genes of microbial origin. Key residues previously proposed to have roles in substrate binding and catalysis in sheep 6‐phosphogluconate dehydrogenase are highly conserved in this entire family of enzymes. Site‐directed mutagenesis, chemical modification, and substrate specificity studies were used to compare possible mechanistic similarities of 3‐hydroxyisobutyrate dehydrogenase with 6‐phosphogluconate dehydrogenase. The data suggest that 3‐hydroxyisobutyrate and 6‐phosphogluconate dehydrogenases may comprise, in part, a previously unrecognized family of 3‐hydroxyacid dehydrogenases.

Are aromatic residues essential at the “active sites” of peptide hormones?

Abstract We have carried out a detailed analysis of the region of the hormone, which can be called an “active site”, that is essential for receptor binding and/or agonist activity, on the basis of structure-activity data of peptide hormones published in the literature. We find that one or more aromatic residues, often in a cluster, is present at the active sites of insulin, glucagon, adrenocorticotropin, gastrin, endorphins and angiotensin. Recognition of the functional importance of aromatic residues, combined with sequence comparisons and secondary structural predictions, enable us to identif active sites of nerve growth factor, somatostatin, calcitonin, parathyroid hormone and luteinizing hormone releasing hormone. Aromatic residues also appear to be essential for the function of nonpeptide hormones that act at the plasma membrane, and opiates. The apparently ubiquitous presence of aromatic groups at the active site of peptide hormones may facilitate understanding of the mechanism of action of hormones and provide insights into the design of hormone analogues.

Kinetics of the two-sited enzyme. II. A method of distinguishing between anticooperative and independent active sites based on competitive inhibition.

Abstract The presence of two active sites on an enzyme leads to downwardly curving Lineweaver-Burk plots if (A) the sites are independent, but have different Michaelis constants, or (B) if the sites interact anticooperatively to impair binding, but not catalysis, at the second site filled. Cases A and B are kinetically indistinguishable when only enzyme and substrate are present. However, equations derived by the rapid-equilibrium treatment show that the two cases have different patterns of competitive inhibition and become distinguishable in the presence of a suitable inhibitor. The inhibitor may decrease or increase the curvature of Lineweaver-Burk plots, but certain patterns have diagnostic value because they can occur only in case (B). In one type of diagnostic pattern, high concentrations of inhibitor cause the Lineweaver-Burk plots to curve upward, and cause the corresponding saturation curves to become sigmoid. The effect of the inhibitor is thus to make sites which are anticooperative appear to be cooperative. This suggests that the mere occurrence of sigmoid saturation curves is not necessarily evidence of cooperative binding effects, and may have uncertain significance in considerations of enzyme regulation.

CONVENIENT SYNTHESIS OF BENZOYLECGONINE ETHYL ESTER, A HOMOLOG OF COCAINE

Abstract Benzoylecgonine reacted with tetramethylethylenediamine to form a lipophilic ion pair, which was alkylated in the absence of water. The ethyl ester was readily recrystallized for pharmacological studies.

Slow reversible inhibition of rabbit muscle aldolase by D-erythrulose 1-phosphate

Rabbit muscle aldolase was found to be inactivated in a slow, reversible manner by D-erythrulose 1-phosphate. This compound combined rapidly and reversibly with the enzyme to form an initial complex, which then only slowly (ki = 0.28 min-1) converted to a kinetically more stable form. This stable enzyme-ligand form was inactive toward the normal substrate of aldolase, fructose 1,6-bisphosphate. The inactive enzyme-ligand complex, however, could be decomposed (kr = 0.0041 min-1) to yield active enzyme once again by incubation in a solution devoid of D-erythrulose 1-phosphate.

Structural and Mechanistic Aspects of a New Family of Dehydrogenases, the β-Hydroxyacid Dehydrogenases

The catabolism of valine, unlike that of other branched-chain amino acids, occurs with the formation of a free branched-chain acid, (S)-β-hydroxyisobutyrate or HIBA, whereas other branched-chain amino acids are metabolized solely as coenzyme A thioesters. Because it exists as a free acid, HIBA can be released into the blood stream by specific tissues and is cleared by the liver where it can serve as a substrate for gluconeogenesis (Letto et al., 1986). During the past decade there has been significant interest in the metabolism and interorgan trafficking of the R- and S- enantiomers of HIBA. HIBA is oxidized in mitochondria to methylmalonate semialdehyde by a highly specific, NAD+-dependent dehydrogenase (HIBADH or 3-hydroxy-2-methyl-propionate: NAD+ oxidoreductase, EC 1.1.1.31). Previous studies of rat HIBADH tentatively placed the enzyme in the now well-established short-chain alcohol dehydrogenase family (Crabb et al., 1993; Hawes et al., 1995). This assignment was based on amino acid sequence homology, enzymatic properties such as the lack of a metal requirement for catalysis, and effects of tyrosine-specific chemical modification. However, site-directed mutagenesis studies indicated that HIBADH differs in mechanism from the short-chain dehydrogenases studied to date, such as Drosophila alcohol dehydrogenase (Hawes et al., 1995). Furthermore, the short-chain dehydrogenases mostly prefer secondary alcohols as optimal substrates whereas HIBADH is only active with primary alcohol substrates. HIBADH, therefore, is most likely not closely related to the short-chain dehydrogenases. More recent studies showed that HIBADH shares better amino acid sequence homology and enzymatic properties with a separate, previously unrecognized family of enzymes that includes D-phenylserine dehydrogenase from Pseudomonas syringae, 6-phosphogluconate dehydrogenase from numerous species, and numerous hypothetical proteins of microbial origin (Hawes et al., 1996).

Student‐as‐Scientist and scientist‐as‐Student: Changing Models for Learning From Experience

1We would like to thank the reviewers for suggesting KidsNetwork as an appropriate example of active involvement and Audrey B. Campaign for reminding us to include the social nature of the scientific enterprise.

Ion-Affinity Electrophoresis. Preparation and Properties of Phosphorylated Polyacrylamede Gels

Strong, pliable polyacrylamide gels containing covalently bound phosphate groups have been formed by radical-initiated copolymerization of acrylamide and a phosphorylated, N-substituted derivative of acrylamide. Under conditions of disc electrophoresis, the gels provide enhanced resolution in the separation of hemoglobin A from S, and separate two forms of yeast glucose 6-phosphate dehydrogenase and rabbit muscle glyceraldehyde 3-phosphate dehydrogenase. Preliminary evidence suggests that the improved resolution is due to ionic adsorption of the proteins to immobilized phosphate groups, implying that the gels may function as supports for electrophoretically powered separations involving ion-exchange or affinity chromatography.