Studies on the lipid composition of the rat liver endoplasmic reticulum after induction with phenobarbitone and 20-methylcholanthrene

1. The cholesterol content, proportions of different phospholipids and fatty acid components of phosphatidylcholine and phosphatidylethanolamine were studied in rat liver endoplasmic-reticulum membrane, after a single injection of 20-methylcholanthrene or injections of phenobarbitone for 5 days. 2. A marked decrease in the proportion of cholesterol occurred 5 days after injection of 20-methylcholanthrene or phenobarbitone. 3. The proportion of phosphatidylcholine was increased by injection of phenobarbitone and minor changes occurred in other phospholipids. 4. Phenobarbitone caused the proportion of linoleic acid in phosphatidylcholine and phosphatidylethanolamine to increase to 120-125% of the control and the proportion of oleic acid, arachidonic acid and docosahexaenoic acid to decrease. 5. 20-Methylcholanthrene caused an increase in the proportion of oleic acid in phosphatidylcholine and ethanolamine to 125-140% of the control, 1 day after injection. 6. The increased proportion of linoleic acid in phosphatidylcholine after phenobarbitone injection occurs simultaneously with the increase of cytochrome P-450 concentration, the rate of oxidative demethylation of aminopyrine and the rate of hydroxylation of biphenyl. It is therefore considered that distinct species of phosphatidylcholine or phosphatidylethanolamine containing linoleic acid in the beta position are essential in the endoplasmic-reticulum membrane for optimal activity of oxidative demethylation.     

Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S(0)]/v(i) (initial substrate concn./initial velocity) versus [S(0)] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S(0)]/v(i) versus [S(0)] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S(0)]/v(i) versus [S(0)] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25 degrees C is characterized by K(m) (1) (the dissociation constant of ES)=1.22+/-0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57x10(-2)+/-0.32x10(-2)s(-1), K(a) (2) (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38+/-0.06m, and k' (the rate constant for the breakdown of SES to E+P+S)=0.45+/-0.04s(-1). 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester and of alpha-N-benzoyl-l-arginine amide; K(m) (1) and k for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine amide hydrolysis and K(as) and k' for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of k(cat.) and K(m) for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (K(m)=52+/-4mm, k(cat.)=2.80+/-0.1s(-1) at pH7.0, I=0.1, 25.0 degrees C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.     

The use of deoxyfluoro-d-galactopyranoses in a study of yeast galactokinase specificity

1. 2-Deoxy-2-fluoro-d-galactose, 3-deoxy-3-fluoro-d-galactose, 4-deoxy-4-fluoro-d-galactose, 6-deoxy-6-fluoro-d-galactose and 2-deoxy-d-lyxo-hexose are substrates for yeast galactokinase. 2. The variation in K(m) values for the d-hexose derivatives was not associated with a variation in the value of K(m) for MgATP(2-) indicating that the binding of MgATP(2-) is not modified by the binding of the sugar substrate. 3. Donated H bonds from OH-3, OH-4 and OH-6 and an accepted H bond to OH-2 of the d-hexose are important for the binding of the sugar substrate to galactokinase. 4. Yeast galactokinase exhibits similar kinetics to the galactokinase from Escherichia coli and operates by a similar random sequential mechanism. 5. 4-Deoxy-4-fluoro-d-glucose was neither a substrate for nor an inhibitor of yeast galactokinase.     

Intra- and extraganglionic peripheral cholinergic neurons in the urogenital organs of the cat

Summary The distribution of cholinergic neurons in the urinary tract and male genital organs of the cat was studied by a histochemical method for acetylcholinesterase. In addition to cell clusters in autonomic ganglia (intraganglionic cells), isolated extraganglionic cholinergic cells were found within the innervated tissues, usually in association with nerve trunks and blood vessels. Smaller neural cells with multiple axonal processes, identical to Cajal's interstitial cells, were found in the meshes of the terminal nerve plexus in smooth muscle, lamina propria and vascular wall.It is concluded that peripheral cholinergic neurons, like their adrenergic analogues, are arranged as a short intraganglionic, a shorter extraganglionic, and a terminal system of neurons.Supported in part by grants 10465 and 11285 from the USPHS and the Henry C. Buswell Urology Research Fund.     

Computed Action Potentials for Purkinje Fiber Membranes with Resistance and Capacitance in Series

The Hodgkin-Huxley equations, as modified by Noble for computation of Purkinje fiber action potentials, have been solved numerically for a membrane whose equivalent circuit contains a constant resistance in series with part of the capacitance. The rates of depolarization and repolarization of the computed action potential have thereby been brought into agreement with measured values. Possible explanations of the frequently observed pre-plateau notch and of fibrillatory activity arise. The effects of a time-dependent K conductance dependent on the second power of the parameter n, instead of the fourth, have also been considered.