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D Zakim

Publications and source records attributed to D Zakim.

At least 91 records · Page 5Linked to original sources
PubMed1976

Regulation of microsomal UDP-glucuronyltransferase by metal ions. Differential effects of Mn2+ on forward and reverse reactions.

Mn2+ activates UDP-glucuronyltransferase (assayed with p-nitrophenol as aglycone) in assays of the forward reaction. The kinetic mechanism of this activation is an increase in activity at V. The rate of the reverse reaction catalyzed by UDP-glucuronyltransferase (UDP-dependent hydrolysis of p-nitrophenylglucuronic acid) is inhibited by Mn2+. The basis for this inhibition is an increase in KUDP, and a small increase in KNphGlcUA. Mn2+ appears to have no effect of the activity of the reverse reaction of saturating concentrations of both substrates. The differential effects of divalent metal ions on rates of the forward and reverse reactions catalyzed by UDP-glucuronyltransferase are due in part to differences in the affinities of UDP-glucuronic acid and UDP for metals. Keq for the formation of UDP-Mn2+ is 8.6 X 10(3); Keq for the formation of UDP-glucuronic acid-Mn2+ is 4.8 X 10(2). High concentrations of UDP thus can compete for metal ions bound to UDP-glucuronyltransferase, but UDP-glucuronic acid is less likely to do so. This competition for metal ions between the enzyme and substrates accounts for the failure of Mn2+ to increase the activity at V of the reverse reaction. Mn2+-induced inhibition of the reverse reaction, due to an increase of KUDP, reflects that the affinity of the affinity of the enzyme for UDP is greater than that for UDP-Mn2+. On the other hand, formation of a complex between UDP-glucuronic and Mn2+ does not alter the affinity of UDP-glucuronyltransferase for this nucleotide derivative

Animals↗
PubMed1976

Evidence for multiple enzymes in the dolichol utilizing pathway of glycoprotein biosynthesis.

A comparison has been made of the enzymes catalyzing the transfer of mannose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented with suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not the reverse of the N-acetylglucosamine or mannose trasfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.

Animals↗
PubMed1976

Defective function of a microsomal UDP-glucuronyltransferase in Gunn rats.

The kinetic parameters of the p-nitrophenol-metabolizing form of UDP-glucuronyltransferase [-UDPglucuronosyltransferase; UDPglucuronate beta-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17] have been compared in liver microsomes from the Gunn strain of rat and from normal; Wistar rats. The abnormally low rate of glucuronidation of p-nitrophenol in the Gunn rats, as compared with Wistar rats, is due to decreased affinity of UDP-glucuronyltransferase for UDP-glucuronic acid. Activities at Vmax and the Michaelis constant for p-nitrophenol, KPNP, of UDP-glucuronyltransferase are the same for enzyme from either strain of rat. Studies of the kinetic parameters of the reverse reaction catalyzed by UDP-glucuronyltransferase indicate that the enzyme from Gunn rats also has decreased affinity for UDP. Calculated values of deltaG degrees for the binding of the UDP portion of UDP-glucuronic acid suggest that the defect of UDP-glucuronyltransferase of Gunn rats appears limited to abnormal interactions between the enzyme and the UDP portion of UDP-glucuronic acid. Studies of the extent of UDP-induced inhibition of the forward reaction support this idea. Diethylnitrosamine, added to microsomes in vitro, enhances the affinity of UDP-glucuronyltransferase for the UDP portion of UDP-glucuronic acid. Despite the defective conformation of the UDP-glucuronic acid binding site of UDP-glucuronyltransferase from Gunn rats this enzyme is activated in the normal way by UDP-N-acetylglucosamine, which is a K-type effector with regard to UDP-glucuronic acid.

Allosteric Regulation↗
PubMed1976

Studies on serum amylase in normal man and in acute pancreatitis.

Amylase isoenzymes in serum, urine, saliva, jejunal juice, and pancreatic tissue were separated by isoelectric focusing. Isoamylase patterns obtained indicated that the majority of amylase activity in normal serum is of salivary gland origin. Pancreatic amylase is characteristically predominant in acute pancreatitis. The increased renal clearance of amylase in acute pancreatitis may be partly due to the increased proportion of the smaller molecular weight pancreatic amylase. However, a demonstrated increase in the renal clearance of salivary amylase in acute pancreatitis suggests a renal cause also. Autopsy pancreas samples devoid of TAME (p-tosyl arginine methyl ester) esterase activity (e.g. trypsin and plasma enzymes such as thrombin and plasmin) had isoenzyme patterns different to those samples with free proteolytic activity. Incubation of TAME esterase free pancreas with trypsin caused conversion of the former isoamylase pattern to one with the predominant isoenzymes focusing coincident with the predominant peak in serum from acute pancreatitis, jejunal aspirate, and TAME esterase positive autopsy pancreas. Such conversion suggests that pancreatic amylase is synthesized in a form different from that found in the intestinal lumen and serum.

Acute Disease↗
PubMed1975

The transfer of galactose from UDP-galactose to endogenous lipid acceptors in liver microsomes.

When the microsomal fraction of beef liver is incubated with UDP-[14-C]-galactose in the presence of an inhibitor of nucleotide pyrophosphatase, there is an incorporation of the [14-C]galactose into glycoprotein and into two lipid components, one soluble in chloroform and the other in chloroform/methanol/water (1:1:0.3). Chromatography of the chloroform fraction on DEAE-cellulose or Kieselguhr G gives a single peak with behavior identical to that of dolichol phosphate mannose. Hydrolysis of the chloroform fraction released free galactose. It seems, therefore, that galactose, like glucose, mannose, and N-acetylglucosamine, can be transferred from its respective sugar nucleotide to glycoprotein via dolichol intermediates.

Adenosine Triphosphate↗
PubMed1975

Characterization of the reaction of GDP-mannose with dolichol phosphate in liver membranes.

The Mn-2+ dependent mannosyl transfer reaction between GDP-[14-C]mannose and dolichol phosphate, which is catalyzed by liver membranes, could not be followed accurately with the existing assay systems. Thus, GDP-[14-C]mannose is hydrolyzed rapidly by a pyrophosphatase present in microsomal and Golgi fractions from liver cells. The rate of the hydrolysis is rapid enough to limit the extent of incorporation of [14-c]mannose into endogenous acceptors. AMP was an effective inhibitor of the pyrophosphatase in Golgi membranes, and protected GDP-mannose from metabolism in alternative pathways. In the presence of AMP it was possible accurately to follow the time course of synthesis of dolichol phosphate [14-c]mannose over short time periods. Even though the time course of the reaction was measured over 2 s intervals, no linear portion could be detected in plots of product formed versus time. The kinetics of synthesis did, however, fit an equation for a first-order kinetic process. The basis for the first-order kinetics seems related to the very small amounts of dolichol phosphate in membranes. The values of the first-order rate constant is dependent on the concentrations of GDP-mannose and Mn-2+ added to the assays.

Adenosine Monophosphate↗
PubMed1975

The effect of a temperature-induced phase change within membrane lipids on the regulatory properties of microsomal uridine diphosphate glucuronyltransferase.

UDP-glucose, UDP-mannose, and UDP-xylose inhibit microsomal UDP-glucuronyltransferase in assays at 6 degrees. None of these compounds affects activity in assays at 37 degrees. Studies with UDP-glucose revealed an abrupt temperature-dependent change in the effect of this compound on the activity of UDP-glucuronyltransferase. Inhibition by UDP-glucose was observed only at temperatures below 16 degrees. UDP-glucose had no effect on activity in assays at temperatures of 16 degrees and above. UDP-N-acetylglucosamine activates UDP-glucuronyltransferase at temperatures at or above 16 degrees, but has no effect on activity below 16 degrees. The lipid portion of the microsomal membrane is known to undergo a phase change at 16 degrees. These data indicate, therefore, that significant alterations in the regulatory properties of UDP-glucuronyltransferase are associated with temperature-induced phase separations within the lipid portion of the microsomal membrane.

Animals↗
PubMed1974

Stimulation of microsomal uridine diphosphate glucuronyltransferase by glucuronic acid derivatives.

The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3beta-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.

Animals↗