The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme

Abstract

More than 80% of the phospholipid component of guinea-pig liver microsomal membranes (prepared with 154mM-KCl) was removed by treatment with phospholipase A followed by extraction of the lysophosphatides and fatty acids produced with albumin. Delipidation strongly inactivated the highly active UDP-glucuronyltransferase of these preparations and activity was restored by mixtures of phosphatidylcholine and lysophosphatidylchlone. However, small quantities of lysophosphatides were still associated with the delipidated fractions after extraction with albumin and might have influenced the inactivation and re-activation observed. To eliminate these uncertainties, microsomal proteins and phospholipids were separated by gel filtration on Sephadex G-150 in the presence of cholate. This technique also strongly inactivated the enzyme but did not generate membrane-active phospholipid degradation products. High transferase activity was again restored to the delipidated protein by choline glycerophosphatides. These results confirm the view that the fully active form of microsomal UDP-glucuronyltransferase is phospholipid-dependent.

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure

“…Bovine serum albumin (crystalline), lysophosphatidylcholine (egg yolk) and linoleic acid were from Sigma (London) Chemical Co., Kingston upon Thames, Surrey, U.K. The albumin was defatted as described by Graham et al (1977). Purified phospholipase A (proteinase free) was prepared as described by Graham & Wood (1969).…”

“…Liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17) is modulated in two ways by the intact phospholipid structure of native microsomal membranes (Graham et aL, 1974a;Graham et al, 1977). In certain microsomal preparations, especially those isolated with 0.25Msucrose, phospholipids restrict activity, and recent evidence (see Graham et aL, 1977;Wilkinson & Hallinan, 1977) favours the view that transferase molecules are located deep within the microsomal membranes so that phospholipids act as part of a hydrophobic barrier hindering access of reactants to the sites of glucuronidation. Mild perturbation of phospholipid structure probably activates latent UDP-glucuronyltransferase by increasing membrane permeability Graham et al, 1977;Wilkinson & Hallinan, 1977).…”

“…Transferase activity is much less latent when microsomal fractions are isolated and washed with 154nM-KCl, and in guinea-pig microsomal membranes prepared by this method the enzyme is highly active, almost free from latency , 1973) and appears to be located near the outside surface of microsomal vesicles, where it is not greatly affected by permeability T. Pechey & G. C. Wood, unpublished work). In this preparation UDP-glucuronyltransferase requires intact phospholipid for full activity (Graham et al, 1977); degra-Vol. 175 dation of phospholipids or membrane delipidation inactivates this enzyme form and choline phospholipids restore high activity.…”

“…In microsomal fractions containing latent enzyme these dual modulating effects are superimposed, and phospholipid modification causes two opposing effects on the transferase: activation of latent enzyme by disruption of the permeability barrier and inactivation ofenzyme released from latency by destruction of essential phospholipid Graham et al, 1977).…”

Phospholipid-dependence of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase

“…Similarly, a phospholipid requirement has been suggested for UDP-glucuronyltransferase (EC 2.4.1.17) activity. Treatment with phospholipases of microsomal fractions expressing fully active UDP-glucuronyltransferase activity results in a decreased rate of glucuronidation of p-nitrophenol (Graham & Wood, 1969;Atwood et al, 1971 ;Berry et al, 1976Berry et al, , 1978Graham et al, 1977). Phospholipids added to these preparations restored thep-nitrophenol glucuronyltransferase activity.…”