Alcohol and violence in the lives of gang members.

Life within a gang includes two endemic features: violence and alcohol. Yet, to date, most researchers studying gang behavior have focused on violence and its relationship to illicit drugs, largely neglecting the importance of alcohol in gang life. Because alcohol is an integral and regular part of socializing within gang life, drinking works as a social lubricant, or social glue, to maintain not only the cohesion and social solidarity of the gang, but also to affirm masculinity and male togetherness. In addition to its role as a cohesive mechanism, particular drinking styles within gangs may operate, as with other social groups, as a mechanism to maintain group boundaries, thereby demarcating one gang from another. Other examples of internal gang violent activities associated with drinking include fighting between members because of rivalries, tensions, or notions of honor or respect. At a more symbolic level, drinking is associated with two important ritual events in gang life: initiation, or "jumping in,"and funerals. By better understanding the link between drinking and violence among youth gangs, steps can be taken to determine the social processes that occur in the development of violent behavior after drinking.

Immobilization and kinetics of lactate dehydrogenase at a rotating nylon disk.

Lactate dehydrogenase (LDH) was covalently attached to an impervious nylon surface by an improved technique. The procedure allowed the kinetics of the rotating enzyme disk reactor to be successfully explored. This enzyme-disk configuration has potential applications in assays for lactic acid or pyruvic acid in fluids of biological importance (e.g., urine). In order to evaluate and understand the physics and chemistry underlying the kinetics of the heterogeneous biocatalyst, a mathematical model based on the von Karman-Levich theories of rotating electrodes, was developed. It applied well to LDH attached to a disk, under variable NADH concentrations and fixed pyruvic acid. The new theory, leads to the conclusion that the apparent Michaelis constant K(m)(app), varies linearly with f(-1/2), where f is the speed of rotation of the disk. Extrapolation of f(-1/2) to zero gives the Michaelis-Menten constant, K(m), corresponding to the diffusion-free behavior. With immobilized LDH, the diffusion-free K(m) for NADH obtained at 25 degrees C, in phosphate buffer (pH 7.5) using the extrapolation method was 84 muM. This value was in good agreement with the previously published value of 87 muM, obtained with LDH attached to the inner surface of a nylon tubing. However, when compared to the K(m) for a free enzyme system, the 84 muM was about nine times larger, indicating an inherent reduction in the activity of the bound LDH. Since, at extrapolated infinite rotation speeds, diffusion effects were assumed eliminated, the drop in the activity was thought to be due to sterric hinderances imposed on the substrate NADH as a result of having LDH bound to another polymer.

Flow kinetics of immobilized beta-glucosidase.

The enzyme beta-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 degrees C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 degrees C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.

Thermodynamic profiles for alcohol dehydrogenase action in free solution.

Stopped-flow equipment was used to study the kinetics of the reaction between nicotinamide adenine dinucleotide (NAD) and ethanol, catalyzed by yeast alcohol dehydrogenase. By measuring rates over a range of concentrations of NAD and ethanol and of temperatures, thermodynamic profiles were obtained for the reaction, which occurs by an ordered ternary complex mechanism with NAD adding first. There are significant negative entropies of activation and negative entropy changes for the addition of NAD and of ethanol; the breakdown of the ternary complex is, however, accompanied by a positive entropy of activation. The results are consistent with structural constraints associated with the binding of the substrates, these restraints being to some extent removed when the ternary complex undergoes reaction. The system follows a similar pattern to that found with three different varieties of lactate dehydrogenase.

Kinetic equations and mechanisms for activation and inhibition in enzyme systems.

The rates of enzyme reactions that are activated or inhibited by added modifiers can in some cases be expressed as a rational function of the first degree, v = (alpha 0 + alpha 1[Q] )/(beta 0 + beta 1 [Q] ) where [Q] is the concentration of the modifier and alpha 0, alpha 1, beta 0, and beta 1 are functions of rate constants and sometimes of the enzyme and substrate concentrations; the behaviour is then said to be linear. Three simple mechanisms that give rise to linear kinetics are examined, and the conditions under which there is activation or inhibition are determined. Sometimes there is a transition from activation to inhibition as the substrate concentration is varied. Definitions of competitive, uncompetitive, and noncompetitive activation are suggested, by analogy with the generally accepted definitions for inhibition. In second-degree activation or inhibition the rate can be expressed as the ratio of two quadratic polynomials with positive coefficients. Ten patterns are then possible for plots of v against [Q], and they may be classified with respect to (i) overall activation or inhibition, (ii) initial (at [Q] leads to 0) activation or inhibition, (iii) terminal (at [Q] leads to oo) activation or inhibition, and (iv) whether there is an initial inflexion. The general case of an n:n rational function is also discussed.

Stopped-flow kinetic studies of the cellulase-catalyzed conversion of cellulose to glucose.

The kinetics of the cellulase-catalyzed conversion of soluble cellulose into glucose have been studied over a range of substrate concentrations and temperatures, and at pH values ranging from 4.75 to 7.0. Lineweaver-Burk plots were linear and led to V = 6.2muM/s and K(m) = 13.1 mM at pH 5.8 and 25.0 degrees C. The pK values corresponding to the free enzyme are 4.8 and 6.8 and are consistent with carboxyl and imidazole groups as the active ionizing species. These pK values were little changed in the enzyme-substrate intermediate that reacts in the ratedetermining step, suggesting that the ionizing groups are still free in this intermediate. The activation energy corresponding to V/K(m) is 80.6 kJ/mol, and that corresponding to V is 38.7 kJ/mol. The corresponding entropies of activation are 21 J K(-1) mol(-1) and -157 J K(-1) mol(-1), respectively.

Kinetics of yeast alcohol dehydrogenase and its coenzyme coimmobilized in a tubular flow reactor.

Yeast alcohol dehydrogenase and nicotinamide adenine dinucleotide (NAD) were coimmobilized, with covalent attachment, to the interior surface of a nylon tube. The NAD was attahed at the N(6) group of the adenine moiety; an NAD derivative was prepared and attached to free carboxyl groups at a partially hydrolyzed nylon surface. The enzyme was attached, through glutaraldehyde residues, to free amino groups on the surface. Kinetic studies were carried out in which the reduced NAD was recycled by means of phenazine ethosulfate and 2,6-dichlorophenol indophenol. The reaction was studied over a range of flow rates and ethanol concentrations. The variation of rate with flow rate suggested that there was little diffusion control with respect to ethanol and that there was no observable inhibition by the reaction products. These conclusions were confirmed by evidence based on dimensionless parameters for the reaction and by direct inhibition experiments. The apparent Michaelis constant was lower than when only the enzyme was immobilized, suggesting that the immobilized enzyme-coenzyme system is of high efficiency. Overall rates of reaction were lower than when there was saturation with NAD. The tube showed no measurable loss of catalytic activity over a period of one month.

pH dependence of free and immobilized yeast alcohol dehydrogenase kinetics.

A study was made of the influence of pH on the reaction between NAD and ethanol, catalyzed by yeast alcohol dehydrogenase, both in free solution and attached to the inner surface of a nylon tube. A new least-squares analysis of the results has been devised; it is simpler to apply and is more realistic than those previously employed. Analysis of the results for the free enzyme indicated that the free enzyme has two active ionizing groups having pK values of about 6.6 and 8.8. These pK values undergo only small changes when the enzyme is bound to NAD and when it is bound to both NAD and ethanol. With the immobilized enzyme and saturating concentrations of ethanol the rates went through a maximum as the pH was varied from 6.5 to 10.0. With saturating concentrations of NAD there was a steady increase in rate, with no falling off at pH 10. Immobilization generally brought about an increase in the pK values. These increases are attributed partly to a residual negative surface charge which attracts the leaving H+ ions. They are also attributed partly to the formation in the reaction of H+ ions, which cause the local pH to be lower than that in the bulk solution. This effect is more important with saturating NAD ions, since the buffer anions will then be less mobile and less able to mediate the movement of protons.

Flow kinetics of yeast alcohol dehydrogenase attached to nylon tubing.

Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was attached covalently to the inner surface of nylon tubing, and the immobilized enzyme retained its activity over a period of months. A study was made of the flow kinetics for the reaction between ethanol and NAD. With the ethanol held at saturating concentrations there was partial diffusion control, the extent decreasing with increasing flow rate and increasing NAD concentration. With the NAD at saturating concentrations there was no appreciable diffusion control. The apparent Michaelis constants varied with flow rate vf, being linear in vf-1/3, and extrapolation to infinite flow rate (vf-1/3 = 0) gave the intrinsic Michaelis constants. The inhibition by products was also studied. The results for both NADH and acetaldehyde showed mixed competitive and non-competitive inhibition, with a preponderance of the former. Acetaldehyde is the stronger inhibitor, and this is consistent with the lack of dissusion control with variable ethanol. Inhibition by acetaldehyde is not affected by flow rate, but inhibition by NADH is affected, presumably because of the greater degree of diffusion control with variable NAD.

Temperature and pH effects on immobilized lactate dehydrogenase kinetics.

Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing, and kinetic measurements made. The results were interpreted on the basis of the Kobayashi-Laidler treatment of immobilized enzymes in flow systems, various tests being applied to determine the degree of diffusion control. It was established in various ways that the degree of diffusion control increases with (a) decrease in flow rate, (b) decrease in substrate concentration, and (c) decrease in temperature. A number of quantitative relationships, predicted by the theory, were obeyed by the results, for example: (a) Km(app) varies linearly with vf-1/3, where vf is the flow rate, (b) the logarithm of the product concentration at the exit varies linearly with the logarithm of the flow rate, and (c) absolute calculations of product concentrations are in reasonable agreement with experiment. A value of 5 kcal . mol-1 is estimated for the activation energy of the diffusion processes, and of 1 kcal . mol-1 for the chemical processes. When the pH is varied the rates pass through a flat maximum, the pH dependence being less than with the free enzyme.

Kinetics of acetylcholinesterase immobilized on polyethylene tubing.

Acetylcholinesterase was covalently attached to the inner surface of polyethylene tubing. Initial oxidation generated surface carboxylic groups which, on reaction with thionyl chloride, produced acid chloride groups; these were caused to react with excess ethylenediamine. The amino groups on the surface were linked to glutaraldehyde, and acetylcholinesterase was then attached to the surface. Various kinetic tests showed the catalysis of the hydrolysis of acetylthiocholine iodide to be diffusion controlled. The apparent Michaelis constants were strongly dependent on flow rate and were much larger than the value for the free enzyme. Rate measurements over the temperature range 6-42 degrees C showed changes in activation energies consistent with diffusion control.

The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching.

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.

Flow kinetics of lactate dehydrogenase chemically attached to nylon tubing.

Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing; a modified technique, involving benzidine and glutaraldehyde, was used, and the resulting immobilized enzyme showed no loss of activity over a period of several months. An experimental study was made of the flow kinetics for the reaction between pyruvate and reduced nicotinamide adenine dinucleotide in two limiting cases, one substrate in excess and the concentration of the other one varied. A range of flow rates and temperatures was covered. The results were analyzed in various ways on the basis of the Kobayashi--Laidler treatment of flow systems. It was concluded that the kinetics are largely diffusion-controlled, especially at the lower substrate concentrations and flow rates. The values of the apparent Michaelis constants vary with flow rate vf, being linear in vf-1/3, and the values extrapolated to infinite flow rate (vf-1/3 = 0) approach the values for the enzyme in free solution. Analysis of the rates led to activation energies for the diffusion of the two substrates.

Temperature and pH effects with immobilized electric eel acetylcholinesterase.

Kinetic studies were made with 2 forms of immobilized acetylcholinesterase: enzyme trapped in polyacrylamide gel which was cut into slices; and enzyme attached to the inner surface of nylon tubing. Rates were measured at substrate concentrations which were low and high with reference to the Michaelis constant, and over the temperature range 16-40 degrees C. Low activation energies (1.7-2.7 kcal mol-1) were obtained at low substrate concentrations, indicating diffusion control. At high substrate concentrations the Arrhenius plots were non-linear and the activation energies substantially higher, and there is less diffusion control. With enzyme-polyacrylamide slices, there was a continuous increase in rate with increasing pH, in contrast to the bell-shaped behavior with free enzyme. A theoretical treatment suggests that this is due to the lowering of local pH as a result of the acid released in the hydrolysis.

Four-and five-step kinetic models of lactate dehydrogenase.

A five-step model for the reaction catalyzed by beef heart lactate dehydrogenase (EC 1.1.1.27) reconciles differences observed in the four-step model if pre-steady-state data in the forward direction are compared with similar data in the reverse direction. The relationship between the four-and five-step models indicates what problems can develop when an incomplete model is proposed. Nevertheless, there are advantages to using the less complicated four-step model when comparing the molecular kinetics of enzymes catalyzing the same reaction but obtained from different sources.