A study was implemented to quantitate the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) with lactose as the substrate and to investigate various factors which affect these activities. At low lactose concentrations the rate of galactose production was equal to the rate of glucose production. The rate of galactose production relative to glucose, however, dropped dramatically at lactose concentrations higher than 0.05 M and production of trisaccharides and tetrasaccharides began (galactose/glucose ratios of about 2:1 and 3:1, respectively, were found for these two types of oligosaccharides). At least five different trissacharides were formed and their patterns of formation showed that they probably utilized both lactose and allolactose as galactosyl acceptors. Allolactose was produced in amounts proportional to glucose at all lactose concentrations (ratios of allolactose/glucose were about 0.88). Analyses of various data, including a reaction analyzed at very early times, showed that the major means of production of allolactose (and the only means initially) was the direct enzymatic transfer of galactose from the 4 position to the 6 position of the glucose moiety of lactose without prior release of glucose from the enzyme. It was shown, however, that allolactose could also be formed in significant quantities by the transfer of galactose to the 6 position of free glucose, and also by hydrolysis of preformed trisaccharide. A mechanism which fits the initial velocity data was proposed in which the steps involving the formation of an enzyme-gallactose-glucose complex, the formation and breakage of allolactose on the enzyme, and the release of glucose all seem to be of roughly equal magnitude and rate determining. Various factors affected the amounts of transgalactosylase and hydrolase activities occurring. At high pH values (greater than 7.8) the transgalactosylase/hydrolyase activity ratio increased dramatically while it decreased at low pH values (less than 6.0). At mid pH values the ratio was essentially constant. The absence of Mg2+ caused a large decrease in the transgalactosylase/hydrolase activity ratio while the absence of all but traces of Na+ or K+ had no effect. The anomeric configuration of lactose altered the transgalactosylase/hydrolase activity ratios, alpha-Lactose resulted in a decrease of allolactose production (transgalactosylase activity) relative to hydrolase activities (glucose production) while beta-lactose had the opposite effect.

Carrier-mediated transport of drugs occurs in various tissues in the body and may largely affect the rate of distribution and elimination. Saturable translocation mechanisms allowing competitive interactions have been identified in the kidneys (tubular secretion), mucosal cells in the gut (intestinal absorption and secretion), choroid plexus (removal of drug from the cerebrospinal fluid), and liver (hepatobiliary excretion). Drugs with quaternary and tertiary amine groups represent the large category of organic cations that can be transported via such mechanisms. The hepatic and to a lesser extent the intestinal cation carrier systems preferentially recognize relatively large molecular weight amphipathic compounds. In the case of multivalent cationic drugs, efficient transport only occurs if large hydrophobic ring structures provide a sufficient lipophilicity-hydrophilicity balance within the drug molecule. At least two separate carrier systems for hepatic uptake of organic cations have been identified through kinetic and photoaffinity labeling studies. In addition absorptive endocytosis may play a role that along with proton-antiport systems and membrane potential driven transport may lead to intracellular sequestration in lysosomes and mitochondria. Concentration gradients of inorganic ions may represent the driving forces for hepatic uptake and biliary excretion of drugs. Recent studies that aim to the identification of potential membrane carrier proteins indicate multiple carriers for organic anions, cations, and uncharged compounds with molecular weights around 50,000 Da. They may represent a family of closely related proteins exhibiting overlapping substrate specificity or, alternatively, an aspecific transport system that mediates translocation of various forms of drugs coupled with inorganic ions. Consequently, extensive pharmacokinetic interactions can be anticipated at the level of uptake and secretion of drugs regardless of their charge.

1. The existence of two different ~-glucose-6-phosphate dehydrogenases in Pseudomonas Juorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway.2. A procedure is described for the isolation of that ~-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44 %. The final preparation, having a specific activity of about 300 pmol NADH formed per min per mg protein, was shown to be homogeneous.3. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000.4. The p l of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9.5. The Entner-Doudoroff enzyme showed specificity for NAD' as well as for NADP' and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory.6. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of P-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase.In organisms of the genus Pseudomonas glucose 6-phosphate may be metabolized via the Entner-Doudoroff pathway, which is characterized by the dehydratation of 6-phospho-~-gluconate to 2-keto-3-deoxy-6- phospho-D-gluconate and its subsequent cleavage to pyruvate and ~-glyceraldehyde-3-phosphate [ 1,2]. Although it is the sole pathway of D-glucose 6-phosphate metabolism in Ps. saccharophila, the additional existence of the hexose monophosphate pathway has been demonstrated in other species of Pseudomonads [3 -51. Both these pathways have in common the initial dehydrogenation of D-glucose 6-phosphate at carbon-1 and the subsequent hydrolysis of 6-phospho-~-glucono-l,5-lactone. According to that, the overlapping catabolic Entner-Doudoroff and anabolic hexose monophosphate pathways must be susceptible to metabolic regulation. The activity of ~-glucose-6-phosphate dehydrogenase from a number of Pseudomonads has been demonstrated to be subject to metabolic control by ATP or related triphosphates [6-91. Furthermore in the case of Ps. multivorans two ~-glucose-6-phosphate dehydrogenases have been

1. Photoaffinity labelling of human serum albumin with the sodium salts of (3fl-azido-7a,12~-dihydroxy-5~-cholan-24-oyl)-2-amino[2-3H(N)]ethanesulfonic acid, (7,7-azo-3~,12ct-dihydroxy-5P-cholan-24-0yl)-2-amino[2-~H (N)]ethanesulfonic acid and (1 1 [-azido-12-0~0-3a,7a-dihydroxy-5~-cholan-24-oyI)-2-amin0[2-~H (N)]ethanesulfonic acid resulted, in each case, in a considerable covalent incorporation of radioactivity into the protein.2. Photoaffinity labelling of whole serum, obtained from fasting test persons, revealed with all three photolabile derivatives of taurocholate at the physiological concentration of 2.1 pM the incorporation of radioactivity not only into albumin but also into high-density lipoprotein, as demonstrated by density gradient centrifugation and by immunological characterization.3. The bulk of radioactivity incorporated into high-density lipoprotein by photoaffinity labelling of whole serum was found to have been associated with the lipids. Only l0-200/, of the label was covalently bound to apolipoproteins, predominantly to the apolipoproteins A-I and A-11.4. The interaction of taurocholate with high-density lipoprotein has been confirmed by density gradient centrifugation using I4C-labelled taurocholate. It is assumed that the interaction of taurocholate with high-density lipoprotein is physiologically of significance.

1. Photoaffinity labelling of a subfraction of plasma membranes of rat liver, enriched with sinusoidal surfaces, with the sodium salts of (3~-azido-7a,l2r~-dihydroxy-5~-cholan-24-oyl)-2-amino[2-~H(N)]ethanesulfoni acid, (7,7-azo-3r~,12a-dihydroxy-5~-cholan-24-oyl)-2-amino[2-~H(N)]ethanesulfonic acid and ( 1 I~-azido-12-oxo3r,7a-dihydroxy-5~-cholan-24-oyl)-2-amino[2-3H(N)]ethanesulfonic acid resulted with each derivative in a clear covalent incorporation of radioactivity into polypeptides with the apparent molecular weights of 67 000, 52 000, 48000,43000 and about 20000.2. Photoaffinity labelling of a membrane subfraction predominantly composed of bile canalicular membranes by the photolabile derivatives of the conjugated bile salts also showed covalent incorporation of radioactivity into polypeptides of the same apparent molecular weights as with the subfraction enriched with the sinusoidal membranes.3. The extent of photoaffinity labelling of the different membrane polypeptides is dependent upon the photolabile bile-salt derivative used. However, with each of the photolabile derivatives the relative ratio of the labelling of the different membrane polypeptides was similar for both membrane subfractions. Provided that the uptake as well as the secretion of bile salts by hepatocytes are carrier-mediated processes, this suggests the participation of the same polypeptides in both processes.In the course of enterohepatic circulation, bile salts are taken up by the hepatocyte from portal blood and secreted into the bile. This functional polarity becomes morphologically apparent in a regional specialization of the surface membrane in areas for uptake and for secretion. Both the uptake by the sinusoidal membrane and the secretion by the bile canalicular membrane seem to be carrier-mediated transport processes [I -91, the secretion being the rate-limiting step in the over-all transport from blood to bile [7,8]. Under physiological conditions conjugated and unconjugated bile salts cross the sinusoidal cell membrane. Based on kinetic measurements, more than one carrier for the uptake of bile salts by hepatocytes has been postulated [lo, 111. These different carriers are assumed to be partly specific for the conjugated derivatives and to be partly shared by conjugated and unconjugated bile salts [I I].Most investigations so far have been concerned with the characterization of the kinetics of uptake and secretion processes for bile salts, and only a few studies have been perAbbreviation. Hepes, 4-(2-hydroxyethyI)-l-piperazineethanesulfonic acid.Enzyrnc~.