Allan B. Graham

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.

Urinary excretion of salicyluric and salicylic acids by non-vegetarians, vegetarians, and patients taking low dose aspirin

Aim: To compare amounts of salicyluric acid (SU) and salicylic acid (SA) excreted daily in the urine of non-vegetarians and vegetarians not taking salicylate drugs, and patients taking 75 or 150 mg aspirin/day. Methods: Urine excreted over 24 hours was collected from volunteers in the four groups. The volumes were recorded and the concentrations of SU and SA were determined electrochemically after separation by high performance liquid chromatography. Results: Significantly more SU was excreted daily by vegetarians (median, 11.01; range, 4.98–26.60 μmol/24 hours) than by non-vegetarians (median, 3.91; range, 0.87–12.23 μmol/24 hours), although amounts were significantly lower than those excreted by patients taking aspirin. Median amounts of SU excreted by patients taking 75 and 150 mg/day of low dose aspirin were 170.69 (range, 13.15–377.18) μmol/24 hours and 165.17 (range, 5.61–429.12) μmol/24 hours, respectively. The amount of SU excreted by patients taking either 75 or 150 mg of aspirin/day was not significantly different. Significantly more SA was excreted by vegetarians (median, 1.19; range, 0.02–3.55 μmol/24 hours) than by non-vegetarians (median, 0.31; range, 0.01–2.01 μmol/24 hours). The median amounts of SA excreted by vegetarians and the patients taking aspirin were not significantly different. Conclusions: More SU and SA is excreted in the urine of vegetarians than in non-vegetarians, consistent with the observation that fruits and vegetables are important sources of dietary salicylates. However, significantly less SU was excreted by vegetarians than patients taking aspirin, indicating that the daily intake of bioavailable salicylates by vegetarians is considerably lower than that supplied by a single 75 or 150 mg dose of aspirin.

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.

Identification and determination of salicylic acid and salicyluric acid in urine of people not taking salicylate drugs

Background Salicylic acid (SA) is present in the serum of people who have not taken salicylate drugs. Now we have examined the urine of these subjects and found that it contains SA and salicyluric acid (SU). We have established the identities of these phenolic acids and determined their concentrations. Methods and Results The acidic hydrophobic compounds of urine were separated using high-performance liquid chromatography (HPLC) and were detected and quantified electrochemically. Two approaches were used to establish the identity of SA and SU. First, the retention times (Rt) of the substances extracted and those of SA and SU were compared under two sets of chromatographic conditions; the Rt of the compounds suspected to be SA and SU and those of the authentic substances were very similar under both sets of conditions. Second, the unknown substances, isolated by HPLC, were treated with acetyl chloride in methanol and compared with the methyl esters of SA and SU by using gas chromatography-mass spectrometry; the unknown compounds after esterification had very similar mass spectra and gas chromatographic Rt to those of methyl salicylate and methyl salicylurate. The median (n = 10) urinary concentration of SA was 0·56 μmol/L (range 0·07-0·89 μmol/L) and that of SU was 3·20 μmol/L (range 1·32-6·54 μmol/L). SA and its major urinary metabolite, SU, were found in the urine of all of the 10 people examined.