David Zakim

A spin-label study of the role of phospholipids in the regulation of membrane-bound microsomal enzymes☆

Abstract The activities of two hepatic microsomal enzymes, glucose 6-phosphatase and UDP-glucuronyltransferase, were determined at assay temperatures in the range between 5 °C and 40 °C. Arrhenius plots of the activities of both enzymes display abrupt changes at about 19 °C. An additional discontinuity at 32 °C is observed in the case of UDP-glucuronyltransferase. Only the latter discontinuity is detected in microsomes subjected to partial treatment with phospholipase A. Lipophilic nitroxide radicals were introduced into samples of the same microsomal preparations and the corresponding electron spin resonance spectra were recorded over the same temperature range. Temperature dependence of an empirical spectral parameter, related to the fluidity of the matrix solubilizing the moleoular probes, reveals apparent breaks at 19 °C and 32 °C in intact microsomes. Only the break at 19 °C was observed in microsomes subjected to sonic disruption. No breaks were detected in plots of data measured in microsomes partially treated with phospholipase A. The correlation between the enzymatic data and the data obtained from lipophilic spin-probes is indicative of the dependence of tightly bound membrane enzymes on the physical state of membrane lipids. The relevance of the data to further studies of the protein-lipid interactions is discussed.

Kinetic properties of microsomal UDP-glucuronyltransferase evidence for cooperative kinetics and activation by UDP-N-acetylglucosamine

The kinetics of microsomal UDPglucuronyltransferase (EC 2.4.1.17) with p-nitrophenol and o-aminobenzoate as glucuronyl acceptors deviate from Michaelis-Menten at low concentrations of UDPglucuronic acid in that double reciprocal plots are concave. The deviation from linearity in these plots represents a real property of UPDglucuronyltransferases and does not result from artifacts in the assay due to metabolism of substrates or products in side pathways. Careful analysis of the data indicated that they also cannot be explained by postulating multiple enzymes for the synthesis of each glucuronide. The most reasonable and simplest mechanism which is compatible with all the data is that there is cooperativity in the sequential binding of UDPglucuronic acid to UDPglucuronyltransferase. Thus, the binding of the first molecule of UDPglucuronic acid to the enzyme makes the subsequent binding of UDPglucuronic acid more difficult. Assay of UDPglucuronyltransferase in the presence of UDP-N-acetylglucosamine is associated with a reversible decrease in the apparent K0.5 for UDPglucuronic acid with p-nitrophenol or o-aminobenzoate as aglycone. The binding of UDP-N-acetylglucosamine to UDPglucuronyltransferase also shows anomalous kinetics which appear to be most compatible with negative cooperativity. Despite similarities in their structures there is no overlap in the binding of UDPglucuronic acid and UDP N-acetylglucosamine at the active site and regulatory site of the enzyme.

Structural, functional and hybridization studies of the glutathione S-transferases of rat liver

We have purified five forms of glutathione S-transferase from rat liver. One form was the glutathione S-transferase B (ligandin), which is composed of two non-identical subunits with molecular weights of 22,000 (Ya) and 25,000 (Yc). Two of the other transferases were Ya and Yc homodimers. The other two transferases were also homodimers, but their subunit, Yb, had a molecular weight of 24,000. The three proteins containing either Ya or Yc subunits had similar substrate specificities, and all three contained peroxidase activity. The greatest peroxidase activity was present in proteins containing the Yc subunit. Enzymes composed of Yb subunits had minimal peroxidase activity in addition to different substrate specificities. The Ya and Yc containing enzymes bound the ligands bilirubin, and indocyanine green with high affinity (KD less than 5 microM), although the KD values of the YcYc protein were consistently 4- to 12-fold greater than those of the other two transferases. Studies were performed to define the origins of the various isozymes. There was no evidence for conversion of Yc to either Ya or Yb during storage or under conditions favorable to proteolysis. Hybridization studies were performed under denaturing conditions (6 M guanidine-HCl), and a YaYc hybrid was formed from the YaYa and YcYc proteins. In addition, both YaYa and YcYc hybrids were formed from transferase B. The hybrids were functionally similar to the proteins isolated originally from the liver. Attempts to form a YaYb hybrid from the YbYb and YaYa transferases were unsuccessful. This result is consistent with the lack of this enzyme form in the liver. Glutathione S-transferase B and the Ya and Yc homodimers appeared to be hybrids of common subunits. These three transferases had very similar functional and structural characteristics and differed from the transferases that are composed of Yb subunits.

Differentiation of homologous forms of hepatic microsomal UDP-glucuronyltransferase. I. Evidence for the glucuronidation of o-aminophenol and p-nitrophenol by separate enzymes.

Using kinetic methods it has been possible to show that there are separate forms of UDPglucuronyltransferase (Ec 2.4.1.17) for the synthesis of p-nitrophenylglucuronide and o-aminophenylglucuronide. Thus, p-nitrophenylglucuronide, which is a product inhibitor of its own synthesis, had no effect of the rate of synthesis of o-aminophenylglucuronide; and 4-methylumbelliferylglucuronide and phenylglucuronide were competitive inhibitors of the glucuronidation of o-aminophenol but had no effect on that of p-nitrophenol. Since previous measurements of the rate of glucuronide synthesis as a function of the concentration of UDPglucuronic acid demonstrated that these aglycones did not share a common UDPglucuronic acid binding site, it was concluded that each phenol was metabolized by a separate form of UDPglucuronyltransferase. In order to compare the properties of the two forms of UDPglucuronyltransferases the effect of sulfhydryl group reagents on activities was studied making use of the fact that the p-nitrophenol form of the enzyme is known to have at least three distinctly different sulfhydryl groups. The o-aminophenol form also contained three such groups with nearly identical reactivities as the p-nitrophenol form, indicating close structural similarities between these enzymes. On the other hand, the sulfhydryl groups of the two forms of UDPglucuronyltransferase could be distinguished by their rates of reaction with mersalyl.

Regulation of microsomal enzymes by phospholipids VI. Abnormal enzyme-lipid interactions in liver microsomes from gunn rats

Abstract The rates of synthesis of some glucuronides by liver microsomes from the Gunn strain of rat are abnormally low, but previous investigators of the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase (UDPglucuronate glucuronyltransferase, EC 2.4.1.17) have reported normal levels of activity in these animals. Data presented in this paper indicate, however, that this enzyme is abnormal in Gunn rats. Thus, treatment of liver microsomes from normal Wistar rats with phospholipase A (EC 3.1.1.4) or Triton X-100 increases the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase 10- and 20-fold, respectively, but these agents do not alter activity in microsomes from homozygous Gunn rats. Similarly, phospholipase A and Triton X-100 activate the o-aminophenol and o-aminobenzoate metabolizing forms of UDPglucuronyltransferase in microsomes from normal rats, but are without effect on the enzyme in microsomes from Gunn rats. In contrast, the rates of synthesis of o-aminophenyl- and o-aminobenzoylglucuronides are increased several fold by addition of diethylnitrosamine to microsomes from Gunn rats indicating that the maximum potential activities of UDPglucuronyltransferases are constrained in liver microsomes from both normal and Gunn rats. These data indicate that assays of UDPglucuronyltransferase in native microsomes are not sufficient for delineating the full extent of the defect in the Gunn rat, that there are defects in the function of at least two proteins in liver microsomes from these animals, and that there are abnormal interrelations between some forms of microsomal UDPglucuronyltransferase and their phospholipid environments.

Differentiation of homologous forms of hepatic microsomal UDP-glucuronyltransferase. II. Characterization of the bilirubin conjugating form

Abstract The glucuronidation of bilirubin by UDPglucuronyltransferase (EC 2.4.1.17) was investigated using a kinetic assay. The role of albumin in the assay was studied at concentrations of bilirubin both above and below its limit of solubility. In assays saturated with respect to bilirubin, albumin was almost without affect on initial rates. At concentrations of bilirubin below its limit of solubility, albumin was inhibitory. Thus, the bilirubin-albumin complex is not a substrate for the enzyme. In assays containing saturating concentrations of bilirubin, the rate of glucuronidation was influenced by conditions which “salt in” bilirubin. Using a bisubstrate kinetic analysis, the dissociation constant for the enzyme bilirubin complex was determined to be 12–18 μM. The enzyme was shown to be stimulated by the allosteric effector UDP-N-acetylglucosamine which caused an increase in the apparent affinity of the enzyme for UPDglucuronic acid. The bilirubin conjugating form of UDPglucuronyl-transferase showed the same general behavior toward sulfhydryl reagents and perturbers of the lipid environment as previously studied forms. However, because of differences in the response of the rate of conjugation of bilirubin to metals, UDP-N-acetylglucosamine, and sulfhydryl reagents, it has been concluded that the bilirubin conjugating enzyme differs from the p-nitrophenol, o-aminophenol and o-aminobenzoate forms of UDPglucuronyltransferase.

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.

The lipid intermediates arising during glycoprotein biosynthesis in liver microsomes.

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.

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.

The identification of a unique p-nitrophenol conjugating enzyme in guinea pig liver microsomes

Abstract Guinea pig liver microsomes catalyze the transfer of a galacturonic acid residue from UDPgalacturonic acid to p- nitrophenol . Lineweaver-Burk plots of the rate of the galacturonidation reaction as a function of the concentration of UDPgalacturonic acid, at a fixed concentration of p- nitrophenol , are linear for concentrations of UDPgalacturonic acid greater than 4 mM but non-linear below this concentration. This non-linearity is similar to that seen for the synthesis of p- nitrophenylglucuronide , and suggests that there is negative cooperativity in the binding of UDPgalacturonic acid to the enzyme. Also compatible with the notion of allosterism, UDP -N- acetylglucosamine increases the rate of synthesis of p- nitrophenylgalacturonic acid. Treatment of microsomes with Triton X-100 or phospholipase A increases the rate of synthesis of p- nitrophenylgalacturonic acid but to a lesser extent than the conjugation of p- nitrophenol with UDPglucuronic acid. In addition, the enzyme catalyzing the synthesis of p- nitrophenylgalacturonic acid is activated by mersalyl, maximal activation occurring at 1 mM mersalyl; 5 mM mersalyl inactivates the enzyme completely. The properties of the p- nitrophenylgalacturonide and glucuronide synthesizing reactions are, therefore, similar in many respects. On the other hand, UDPgalacturonic acid is not a product inhibitor of the UDP-dependent hydrolysis of p- nitrophenylglucuronide , and p- nitrophenylglucuronide does not inhibit the reaction of p- nitrophenol with UDPgalacturonic acid. It was concluded that the synthesis of p- nitrophenylglucuronide and -galacturonide are catalyzed by separate enzymes.