Geoffrey C. Wood

The phospholipid-dependence of UDP-glucuronyltransferase

Abstract Evidence is presented indicating that specific degradation of the microsomal phospholipid membrane by phospholipase A or phospholipase C produces a concomitant inactivation of UDP-glucuronyltransferase. This inactivation can be reversed by adding phospholipid micelles. It is therefore considered that the activity of the enzyme depends on phospholipids and hence, probably, on the structural integrity of the microsomal membrane.

The phospholipid dependence of UDP-glucuronyltransferase: Conformation/reactivity studies with purified enzyme

Abstract Highly-active purified UDP-glucuronyltransferase from guinea-pig liver microsomal membranes is associated with phospholipids. Removal of these phospholipids inactivated the transferase and caused profound changes in the enzymes circular dichroism spectrum indicating that its secondary structure was drastically altered. Treatment of the delipidated fraction with phosphatidylcholine restored the enzyme to a much more helical, high reactivity conformation. These results show clearly that an intact phospholipid environment is required to maintain the transferase in a reactive conformation.

Factors affecting the response of microsomal UDP-glucuronyltransferase to membrane perturbants

Abstract Evidence is presented indicating a species difference in the levels of UDP-glucuronyltransferase (EC 2.4.1.17) activity of rat and guinea pig liver microsomal fractions. The specific activities of microsomal fractions prepared in 0.154 M KCl are higher than those of fractions prepared in 0.25 M sucrose. The specific activity of UDPglucuronyltransferase in microsomal fractions and the concentration of UDP-glucuronate during assay influence the enzymes response to membrane perturbation by phospholipase A (EC 3.1.1.4) and detergents.

Studies of the activation of UDP-glucuronyltransferase

Abstract Low concentrations of the detergents Triton X-100 and deoxycholate enhanced the activity of rat liver microsomal p-nitrophenol glucuronyltransferase 3-fold without radically altering the activities of pyrophosphatase or β-glucuronidase. Similar concentrations of the detergents, however, had no significant activating effect on the glucuronyltransferase activity of guinea pig liver microsomal fractions prepared in identical manner. Triton X-100 still activated the rat liver enzyme when competition between glucuronyltransferase and pyrophosphatase for UDP-glucuronate was reduced at a higher concentration of the sugar nucleotide and when β-glucuronidase had been substantially inhibited with saccharo-1,4-lactone. It is concluded that the closely associated pyrophosphatase and β-glucuronidase activities are not involved in the activation of the rat liver enzyme by detergents. The activation of rat liver glucuronyltransferase and the differences between that enzyme and guinea pig liver glucuronyltransferase are discussed.

On the activation of microsomal UDPglucuronyltransferase by phospholipase A

Abstract Activation of rat liver microsomal UDPglucuronyltransferase (EC 2.4.1.17) by phospholipase A (EC 3.1.1.4) was reversed by micellar dispersions of several phospholipids and by serum albumin. When microsomal membranes were treated with the phospholipase in presence of albumin, which binds and removes surface-active phospholipid degradation products (lysophosphatides and unsaturated fatty acids), activation was delayed by albumin at a concentration of 1 mg/mg of microsomal protein and completely prevented by higher concentrations of albumin. The UDPglucuronyltransferase of intact microsomal membranes was activated by lysophosphatidylcholine and unsaturated fatty acids at concentrations which were produced by phospholipase treatment, and the activation by lysophosphatidylcholine most closely resembled that by phospholipase A. It is concluded that the activation of UDPglucuronyltransferase by phospholipase A is due to the detergent activity of phosphilopid hydrolysis products and that the enzyme in intact microsomal membranes probably is not constrained by interaction with a specific phospholipid.

Kinetic studies of latent microsomal UDP-glucuronyltransferases kinetics of glucuronidation in intact and perturbant-treated membranes

Double-reciprocal plots (with UDP-glucuronate as varied substrate) of the rate of glucuronidation of p-nitrophenol by the latent UDP-glucuronyltransferases of intact guinea pig and rat liver microsomal membranes (prepared with 154 mM KCl and 0.25 M sucrose) were continuously curved concave-downwards. Good fits to the kinetic data were obtained by using two different calculation methods which assume that two forms (high K and low K) of the transferase catalyse the reaction simultaneously. No evidence of cooperativity in binding of UDP-glucuronate to the enzyme was found. When latency of the enzymes of these preparations was destroyed by disrupting the membranes with Triton X-100 or lysophosphatidylcholine, double-reciprocal plots were linear. With guinea pig membranes, lysophosphatidylcholine generated an activated single-enzyme form obeying the simple Michaelis-Menten rate law; K for the activated species was close to that (K1) for the native low K form and its value of V was greater than the combined maximum velocities (V1 + V2) of the two forms in intact membranes. With rat membranes, both perturbants produced a single activated form also with V greater than (V1 + V2) and with K2 greater than K greater than K1. These results are discussed and are consistent with the view of transferase latency which envisages that there are two populations (buried and exposed) of enzyme molecules in intact microsomal membranes. The effects of membrane perturbants on the kinetic parameters of the two native transferase forms were assessed by accounting for the possibility that the reactivity of the buried transferase is controlled by the rate of transport of UDP-glucuronate across the membrane matrix. The data are compatible with a model which supposes that UDP-glucuronate gains access to the buried population by a process with the kinetic characteristics of a facilitated transport system.

Purification of granulocyte neutral protease from human blood and rheumatoid synovial fluid

The neutral protease activity of human synovial fluid cells, like that of peripheral blood leucocytes, is located in a granule fraction. It can be solubilised by various agents but only 1 M neutral salts do so without inactivation. Salt-solubilised neutral protease has been purified (300 X) from synovial fluid cells; like preparations obtained in the same way (600 X purified) from peripheral blood leucocytes, it has a broad pH profile of activity (pH 7--10.5) and in this, as well as in substrate specificity and sensitivity to activators and inhibitors, it behaves as a serine-histidine type protease similar to elastase (EC 3.4.21.11). The product showed two major components on polyacrylamide gel electrophoresis. Collagenase or chymotrypsin-like activity were not detected.