Triacylglycerol and very low density lipoprotein secretion into plasma of squirrel monkeys.

We determined the effects of varying the types and level of dietary fat and cholesterol on the increase in plasma total triacylglycerol concentrations after injection of Triton WR-1339, an inhibitor of lipoprotein lipase, into monkeys that had been subjected to an overnight fast. The monkeys that had been treated with Triton WR-1339 were then given a test meal by intragastric intubation. Dietary cholesterol, high levels of fat and saturated fat in the habitual diet reduced the rate of release of triacylglycerol to plasma in the fasted monkey. We also determined the changes in protein and lipid concentrations of the different lipoprotein fractions. The injection of Triton WR-1339 resulted in a linear increase with time in the concentration of protein and triacylglycerol in the very low density (chylomicron-free and d less than 1.006) lipoproteins, but there was an increase in the ratio of traicylglycerol to protein in that fraction. Most of the increase (96%) in very low density protein was in the B protein. Regardless of the habitual diet, a test meal accentuated the rate of triacylglycerol appearance in whole plasma and in the very low density lipoproteins of Triton WR-1339-treated monkeys, and the rate of increase of the protein component after feeding was slightly higher. Thus the administration of a meal to the fasted Triton WR-1339-treated squirrel monkey further increased the proportion of triacylglycerol in very low density lipoproteins. Although dietary cholesterol and saturated fat in the habitual diet depressed the rate of increase in very low density triacylglycerol during fasting, the rate of protein synthesis was not significantly affected. After administration of a test meal the rates of increase in triacylglycerol and protein in the very low density lipoproteins were similar for monkeys from the different diet groups. Triton WR-1339 administration caused a slight and progressive increase in the intermediate density (d 1.006-1.019) lipoproteins and a marked and progressive decrease in the low density (d 1.019-1.063) lipoproteins. There was an immediate (by 5 min) drop of 70% or more in high density (d 1.063-1.21) lipoprotein protein, but the lipids except triacylglycerol remained unchanged. There was a decrease in both the A (the major fraction) and C proteins. The rates of very low density B protein secretion were comparable to the rates of low density lipoprotein catabolism that had been previously demonstrated for this species.     

Some cellular changes associated with triton WR-1339 accumulation in rat hepatocytes.

Electron microscopic morphological and cytochemical techniques were used to follow the sub-cellular events that accompanied Triton WR-1339 accumulation in hepatocytes. Localization of two lysosomal enzymes, acid phosphatase and aryl sulfatase, clearly established the lysosomal nature of the Triton WR-1339 containing electron-lucid structures that appear in hepatocytes following treatment with this compound.     

Hypolipidemic effect and antioxidant activity of glycoprotein isolated from Ulmus davidiana Nakai in Triton WR-1339-treated mouse

The glycoprotein isolated from Ulmus davidiana Nakai (UDN) (UDN glycoprotein) has a molecular weight of 116 kDa and consists of 78.65% carbohydrate content and 21.35% protein content. In the present study, we investigated the hypolipidemic effect of UDN glycoprotein on Triton WR-1339-induced mice. With pretreatment with UDN glycoprotein, the triacylglycerol (TAG), total cholesterol and low density lipoprotein-cholesterol (LDL-C) concentrations were significantly reduced, whereas high density lipoprotein-cholesterol (HDL-C) concentration was increased in the plasma of Triton WR-1339-induced mice. With respect to antioxidative activity, UDN glycoprotein significantly decreased the level of thiobarbituric acid reactive substances (TBARS) and improved activities of catalase and glutathione peroxidase (GPx), without an apparent change of superoxide dismutase (SOD) activity. Also UDN glycoprotein significantly increased nitric oxide (NO) production in Triton WR-1339-induced mice. These results indicate that UDN glycoprotein has a hypolipidemic effect, possesses antioxidant activity and has an ability to stimulate NO production. Thus, we speculate that UDN glycoprotein is an example of natural compound that lowers plasma lipid level together with having an antioxidant function in Triton WR-1339-induced mice.     

Reversal by Triton WR-1339 of ethynyloestradiol-induced hepatic cholesterol esterification

Rats treated with ethynyloestradiol have marked hypolipidaemia: serum cholesterol is decreased to 5%, triacylglycerol to 10% and phospholipid to 70% of control concentrations. Loss of serum cholesterol follows an exponential decay, with a half-life of 1.13±0.09 days. After 4 days of treatment, serum cholesterol concentrations remain relatively constant (ranging from 1 to 20mg/100ml) for at least 30 days. There is a concomitant 20-fold decrease in the d<1.21 fraction of serum proteins and a similar decrease in serum apolipoproteins as measured by sodium dodecyl sulphate/10%-polyacrylamide-gel electrophoresis. The activity of hepatic microsomal acyl-CoA–cholesterol O-acetyltransferase (EC 2.3.1.26) was significantly increased by ethynyloestradiol treatment (P<0.05). This activation caused hepatic cholesteryl esters containing mainly C_18:1 fatty acids to increase linearly as serum cholesterol concentrations decreased (r=0.9675, P<0.001). Triton WR-1339, a non-ionic detergent that inhibits lipoprotein catabolism, was used to estimate hepatic lipid secretion by measuring the increment in serum lipids after its administration. At 15h after Triton WR-1339 administration, serum cholesterol concentrations were increased equally in both control and ethynyloestradiol-treated rats. In contrast, the increment of serum triacylglycerol of treated rats was 40% of that found in control rats, indicating that ethynyloestradiol inhibits hepatic triacylglycerol secretion. Triton WR-1339 inhibited the oestrogen activation of hepatic microsomal acyl-CoA–cholesterol O-acyltransferase and restored hepatic cholesteryl ester concentrations to normal values. These data suggest that ethynyloestradiol and its pharmacological `antagonist' Triton WR-1339 alter hepatic triacylglycerol secretion via a mechanism associated with changes in hepatic cholesterol esterification.     

In vitro effect of Triton WR-1339 on canine plasma high density lipoproteins

We studied the effect in vitro of various concentrations of Triton WR-1339 on normolipidemic canine plasma and on the high density lipoproteins (HDL) isolated from this plasma by ultracentrifugation. As a preamble to this study, we established that Triton WR-1339 has a unimer molecular weight of 4,500, a micellar molecular weight of 180,000, and a critical micellar concentration (CMC) of 0.018 mM or 0.008 g/dl. Above its CMC, Triton WR-1339 in concentrations between 2 and 10 mg/ml induced concentration-dependent structural changes in HDL which were characterized by a progressive displacement of apoA-I from the HDL surface without loss of lipids. The addition of Triton WR-1339 to the HDL particles modified their electrophoresis mobility and caused an increase in size (95 +/- 5 A to 114 +/- 7 A). At the extreme Triton WR-1339 concentrations utilized in these studies (10 mg/ml) disruption of the HDL particles occurred; at this stage, the original, relatively homogeneous, spherical HDL particles were replaced by a heterogeneous population ranging in size between 50 and 250 A, representing complexes of Triton WR-1339 with lipids essentially free of apoA-I which could be sedimented by ultracentrifugation. The effects of Triton WR-1339 on whole plasma or isolated HDL were comparable. These studies indicate that Triton WR-1339 in vitro alters HDL in a concentration-dependent manner and that these changes vary from a displacement of apoA-I from the HDL surface to a state where all lipids are solubilized into the Triton WR-1339 micellar phase and are driven away from the protein moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of lipoprotein lipase activity in cardiac myocytes from control and diabetic rat hearts by plasma lipids.

The objective of this investigation was to test the hypothesis that the diabetes-induced reduction in lipoprotein lipase activity in cardiac myocytes may be due to hypertriglyceridemia. Administration of 4-aminopyrazolopyrimidine (50 mg/kg) to control rats for 24 h reduced plasma triacylglycerol levels and increased the heparin-induced release of lipoprotein lipase into the incubation medium of cardiac myocytes. The acute (3-5 days) induction of diabetes by streptozotocin (100 mg/kg) produced hypertriglyceridemia and reduced heparin-releasable lipoprotein lipase activity in cardiac myocytes. Treatment of diabetic rats with 4-aminopyrazolopyrimidine resulted in a fall in plasma triacylglycerol content and increased heparin-releasable lipoprotein lipase activity. Administration of Triton WR-1339 also resulted in hypertriglyceridemia, but the heparin-induced release of lipoprotein lipase from control cardiac myocytes was not reduced in the absence of lipolysis of triacylglycerol-rich lipoproteins. Treatment with Triton WR-1339 did, however, increase the heparin-induced release of lipoprotein lipase from diabetic cardiac myocytes. Preparation of cardiac myocytes with 0.9 mM oleic acid resulted in a decrease in both total cellular and heparin-releasable lipoprotein lipase activities. These results suggest that the diabetes-induced reduction in heart lipoprotein lipase activity may, at least in part, be due to an inhibitory effect of free fatty acids, derived either from lipoprotein degradation or from adipose tissue lipolysis, on lipoprotein lipase activity in (and (or) release from) cardiac myocytes.     

Effect of triton WR-1339 on peroxisomal enzymes of rat liver

The effect of Triton WR-1339 on peroxisomal enzymes of rat liver was studied. The dose vs. response relationships of peroxisomal enzyme activities to Triton WR-1339 were first examined 3.5 days after injection. Catalase activity was reduced to 50% of that of the control at a dose of 200 mg per 100 g body weight; it was found that the decrease depended on the dose of this compound. Urate oxidase activity was not significantly affected. D-Amino acid oxidase activity showed intermediate behavior. The activities of these enzymes were found to be reduced more markedly at 2 days than at 3.5 days after injection, and subsequently the levels of the activities recovered. At 2 days after injection of a dose of 200 mg per 100 g body weight, the activities of catalase, D-amino acid oxidase and urate oxidase had decreased to 40, 60 and 60%,respectively, of the control values.It was found that the decreases in the activities of these enzymes caused by Triton WR-1339 had occurred in the large granule fraction, but not in the cytoplasm.Measurement of the specific activity, Ouchterlony gel diffusion and quantitative immunoprecipitation suggested that there was a similarity between the Triton WR-1339-treated and untreated rats in the nature of purified catalases.These results suggest that Triton WR-1339 depresses the activities of liver peroxisomal enzymes, especially the catalase activity.     

Effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase of rat.

The effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase was examined. Triton WR-1339 was injected intraperitoneally into rats at a dose of 200 mg per 100 g body weight. Catalase activity decreased to about 35% of that of the control at 42-48 h after the injection and recovered to the normal level at 96 h. Other peroxisomal enzymes, D-amino acid oxidase and urate oxidase, showed similar patterns of the activities to those of catalase. During the first 48 h after the injection of Triton WR-1339, the rate of catalase synthesis (ks) fell to below a detectable value, while that of the degradation (kd) did not show any significant change. On the other hand, during the period 48-96 h after the injection, the rate of the synthesis (ks) returned to the normal level though that of the degradation (kd) decreased to about 50% of the control.     

The effect of Triton WR-1339 on the subcellular distribution of Trypan Blue and 125I-labelled albumin in rat liver

1. The density-gradient distribution patterns of acid phosphatase, Trypan Blue and denatured (125)I-labelled albumin were studied by discontinuous sucrose- and isopycnic sucrose-density-gradient centrifugation on combined heavy and light mitochondrial (M+L) fractions of liver isolated from normal rats and from rats injected with Triton WR-1339. 2. The results obtained from the subfractionation of the M+L pellet of normal animals indicate that the equilibrium density of Trypan Blue and acid-insoluble radioactivity is the same as that for acid phosphatase, which suggests they are bound by a common membrane to form a distinct subcellular population of lysosomal nature. 3. In contrast, the analysis of the isopycnic gradients obtained on subfractionation of M+L pellets of liver isolated from rats treated with Triton WR-1339 show that the acid-insoluble radioactivity has an equilibrium density around 1.21, whereas the acid hydrolases, including cathepsin D, show the characteristic shift to an equilibrium density of around 1.12. Trypan Blue is distributed along the gradient with distinct peaks at densities 1.22 and 1.12. 4. Similar equilibrium-density distribution patterns were obtained with M+L pellets isolated from rats pretreated with Triton WR-1339 but not injected with Trypan Blue. 5. Treatment of the rats with Triton WR-1339 does not affect albumin digestion of isolated intact lysosomes despite the fact that most of the cathepsin D and the albumin ingested by phagocytosis are located in different vacuoles. 6. It is concluded from these experiments that in the liver of animals treated with Triton WR-1339 (125)I-labelled albumin is located within heterophagosomes which do not fuse with heterolysosomes containing the non-ionic detergent Triton WR-1339. The inability of these two lysosomal populations to fuse is not due to Trypan Blue.     

Characterization of rat brain microsomal acyl-coenzyme A ligases: different enzymes for the synthesis of palmitoyl-coenzyme A and lignoceroyl-coenzyme A

Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes.     

Effect of Triton WR-1339 on lecithin-cholesterol acyltransferase in the rat

The effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase was measured in rat serum following addition of Triton to the serum in vitro or after intravenous injection of the detergent. The inhibitory effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase when the detergent was added in vitro was dose dependent and appeared to result from a direct action on the enzyme rather than from a physical modification of the substrate by the detergent. The serum half-life ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of Triton WR-1339 injected intravenously in the rat was 23.1 ± 1.0 h. The inhibitory effect of Triton on serum LCAT activity when the detergent was given intravenously was also dose dependent and was reversed when the serum concentration of Triton decreased; under specific conditions, LCAT activity reached values higher than control. This behavior after treatment of the animal may be explained by increased concentration of the enzyme in the plasma, by stimulation of LCAT activity by the very low density lipoprotein or metabolites accumulating in the plasma of rats treated with Triton WR-1339, or by a combination of these factors.     

Voluntary food consumption and gastric emptying in rats given intravenous Triton WR-1339

Intravenously administered Triton WR-1339, a nonionic surface active agent, has been used as an endogenous hyperlipemic agent since 1951. We expected Triton to increase food consumption to supply, at least partially, the energy and acetyl groups necessary for producing the hyperlipemic state. In this study, however, we observed that the rats injected intravenously with various dose levels of Triton decreased their voluntary food intake in a dose-related manner. Two other nonionic surface active agents, Tween 20 and Tween 80, given intravenously did not alter food intake. Further studies revealed that Triton WR-1339 administered intravenously 30 min before feeding by stomach tube resulted in a marked delay in the rate of gastric emptying which was also dose related. A delay in gastric emptying has previously been suggested as one mechanism that controls food intake. Tween 20 and Tween 80 did not alter the rate of gastric emptying. We suggest that the mechanism responsible for the decrease in voluntary food consumption in Triton WR-1339 injected rats may be due to the delay of gastric emptying in these animals.     

Lysosomes in skeletal muscle tissue. Zonal centrifugation evidence for multiple cellular sources

Postnuclear supernates from homogenates of skeletal muscle from rats subjected to starvation, injections of Triton WR-1339, dextran-500, and dextran + corticosterone were fractionated by means of rate and isopycnic zonal centrifugation in sucrose—0.02 M KCl gradients. Zonal fractions were analyzed for protein, RNA, cytochrome oxidase, and up to six acid hydrolases. The results indicate the presence of two groups of lysosome-like particles. One group contributes approximately 95% of the cathepsin D and acid phosphatase activity and 75% of the acid ribonuclease, β-glucuronidase, and arylsulfatase activity in muscle. It is characterized by a modal equilibrium density of 1.18 that is decreased by starvation, but is not shifted by dextran-500 or Triton WR-1339. The second group has a higher proportion of acid ribonuclease, β-glucuronidase, and arylsulftase; the equilibrium density can be shifted by dextran-500 and Triton WR-1339. It is suggested that this group of lysosomes is derived from macrophages and other connective tissue cells, whereas the former group represents lysosome-like particles from muscle cells.     

Rate of release of hepatic triacylglycerol into serum in the starved rat.

After an intravenous injection of a pulse of [U-14C]palmitate to starved rats, the time-dependent radioactivity profiles were determined in the triacylglycerol (triglyceride) of hepatic microsomal fractions, floating fat, mitochondria and nuclei. The profile of activity in serum gave a value of 0.08 mg/min per 100 g body wt. for the irreversible disposal rate of triacylglycerol from serum. This value, combined with the previously estimated rate of movement of triacylglycerol from serum to liver, and the reported rate from intestine to serum, gave a calculated value of 0.35 mg/min per 100 g body wt. for release rate of triacylglycerol from liver to serum. The rate of release of hepatic triacylglycerol into serum was also measured by the widely used Triton WR-1339 method. The rate obtained with this technique (0.15 mg of triacylglycerol/min per 100 g body wt.) was identical with that reported previously. During the interval from 45 min to 3h after ethanol administration this rate increased to 0.18 mg/min per 100 g body wt. It was concluded that the use of Triton underestimates the true rate of movement of triacylglyerol from liver to serum.     

Ultrastructural studies on the effect of Triton WR-1339 on macrophage-mycobacteria interaction

Mycobacterium bovis (BCG organisms) suspended in saline or a 5% solution of a non-ionic detergent, Triton WR-1339, was injected intraperitoneally into mice. Electron-microscopic observation was carried out on peritoneal exudate cells harvested therefrom. Electron-lucent vacuoles limited by the membrane structure were found in macrophages of the mice injected with BCG suspended in the detergent, but not in polymorphonuclear leukocytes or lymphocytes. Mycobacterial cells were present within such vacuoles. Without the detergent, the ingested mycobacterial cells were in close contact with the phagosomal membrane. Within the electron-lucent vacuoles, however, such close contact was not present. These observations, together with other collateral findings, led us to a view that Triton WR-1339 may inhibit the interaction between mycobacteria and the phagosomal membrane by intervening between them thus making the progress of infection delayed.     

Alpha-tocopherol is secreted from rat liver in very low density lipoproteins

Three separate studies were carried out to test the hypothesis that rat liver secretes vitamin E (alpha-tocopherol) within very low density lipoproteins (VLDL). i) When the clearance of plasma chylomicrons (CM) and VLDL was blocked by the administration of Triton WR-1339, alpha-tocopherol concentrations increased linearly with time in both classes of triacylglycerol-rich lipoproteins, although accumulation rates within VLDL exceeded those within CM. For fasted rats, appearance of alpha-tocopherol in VLDL persisted at slightly reduced rates. alpha-Tocopherol and triglycerides in the VLDL fraction responded to Triton WR-1339 administration by coordinate increases. In contrast to the situation in serum, alpha-tocopherol concentrations decreased in the liver following injection of Triton. ii) In order to inhibit the secretion of hepatic lipoproteins containing apolipoprotein B (apoB), rats were fed a diet containing orotic acid. This resulted in a reduction of apoB and alpha-tocopherol concentrations in serum and VLDL, whereas the vitamin E content of liver was increased. iii) In primary cultures of hepatocytes, alpha-tocopherol was secreted into the culture media predominantly within VLDL. We, therefore, conclude that the liver secretes alpha-tocopherol within VLDL and in this way contributes to the maintenance of serum vitamin E concentrations.     

Intestinal very-low-density lipoprotein secretion in the genetically obese Zucker rat

The objectives of this study were to measure intestinal very-low-density lipoprotein (VLDL) production in obese Zucker rats and to assess an eventual effect of a high-fat diet. VLDL secretion was specifically inhibited by orotic acid, and intestinal VLDL output was measured following the Triton WR-1339 method. After a control diet, total VLDL secretion (without orotic acid) was 4.8 +/- 0.3 and 1.4 +/- 0.1 mg triacylglycerol/ml in obese and lean rats, respectively, decreasing by 30% in obese rats after fat-feeding. Intestinal VLDL production was similar in obese and lean rats fed the control diet (0.32 +/- 0.05 and 0.27 +/- 0.05 mg triacylglycerol/ml, respectively), increasing 2.5-fold after fat-feeding in both genotypes. Thus, intestine contributed 21 and 60% of total VLDL in lean but only 7 and 24% in obese rats with the control and high-fat diets, respectively. These results show that the intestine of obese Zucker rats does not contribute to their hypertriglyceridemia, suggesting that it originates solely from liver. Moreover, their intestinal VLDL production was stimulated by fat-feeding to the same extent as in lean animals.     

The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339.

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

Membranous localization and properties of ATPase of rat liver lysosomes.

Lysosomes isolated from rat liver were found to have ATPase activity (EC No 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2 mM ATP and is inhibited by high concentrations of ATP. The apparent Km for divalent metal is 0.2 mM, and either Ca2+ or Mg2+ give maximal activity. The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity of L fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosaminidase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.