A novel lectin-gold density perturbation eliminates plasma membrane contaminants from Golgi-enriched subcellular fractions

The availability of pure Golgi fractions is a prerequisite for documenting the composition of the membranes of the Golgi Complex and comparing and contrasting this organelle with the rough endoplasmic reticulum. In a companion article, we have described a subcellular fractionation protocol for rat myeloma cells which effectively eliminates rough microsomes from Golgi-enriched fractions. Nevertheless, a major overlap with plasma membrane remains. We have therefore developed a novel density perturbation procedure to eliminate plasma membrane contaminants. By binding a conjugate of wheat germ agglutinin and colloidal gold to cells at 4 degrees C before homogenization we cause extensive sedimentation of plasma membrane markers to the "mitochondrial pellet" as well as a major shift in the isopycnic density of these markers. The differential and isopycnic sedimentation of several Golgi markers is unaffected in lectin-gold treated cells. The Golgi-enriched fractions obtained by isopycnic sedimentation are therefore of high purity. This procedure may be of general use for either purifying or eliminating plasma membrane-derived vesicles. Adaptations of the method might be equally useful for density perturbation of intracellular organelles.     

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.     

Phospholipid and enzyme arrangements of rat liver rough microsomal subfractions from control and methylcholanthrene-treated animals

Rough microsomes from rat liver of both control and methylcholanthrene-treated animals were subfractionated on a discontinuous sucrose gradient into three fractions according the their sedimentation velocity. The slowly sedimenting vesicles were enriched in electron transport enzymes, while those in the pellet showed higher phosphatase and ATPase activities. Methylcholanthrene treatment introduced typical changes in enzyme composition, mainly an increase of the cytochrome P-448. The individual phospholipids exhibited an identical distribution pattern in the three subfractions and no change occurred after induction with methylcholanthrene treatment. Nearest neighbour analysis of phosphatidylethanolamine with dinitrodifluorobenzene revealed a similar pattern in the enzymatically different subfraction, that is, no cross-linking with phosphatidylserine occurred. One-third of the phosphatidylethanolamine was in monomer and dimer form and about two-thirds was protein linked. When membrane and enzyme synthesis was induced, cross-linking to proteins were substantially decreased. The experiments indicate that the phospholipids are distributed in a homogenous fashion in the lateral plane of the rough microsomal membrane and do not support the possibility that phosphatidylethanolamine is specifically associated with cytochrome P-450.     

Subcellular localization of the phospholipases A of rat heart: evidence for a cytosolic phospholipase A1

During myocardial ischemia increased levels of lysoglycerophospholipids have been reported which may be deleterious to myocardial function. Phospholipases are presumed to be important in the regulation of this process. To further quantify and characterize the activity of heart phospholipases, we carried out a systematic analysis of phospholipase A activity in rat heart subcellular fractions isolated by the method of Palmer et al. (J. Biol. Chem. 1972. 262: 8731-8739). Neutral phospholipase A was recovered predominately in the cytosolic (soluble) fraction which represented 46% of recovered activity, while the microsomal and subsarcolemmal mitochondrial fractions represented 15% and 12% of the total recovered activity, respectively. Cytosolic phospholipase A differed from the two principal membrane-bound phospholipases A in its pH dependence and apparent Km for substrate. The cytosolic enzyme had a Km (apparent) for dioleoylphosphatidylcholine of 0.07 mM versus 0.28-0.33 mM for the membrane-associated phospholipases A. Acid phospholipase A activity had a subcellular distribution consistent with a lysosomal localization. Lysophospholipase was found principally in the cytosolic, microsomal, and the subsarcolemmal and interfibrillar mitochondrial fractions where it represented 46, 17, 6.3, and 6.9% of the recovered activity, respectively. The positional specificity of the respective phospholipases was assessed. This analysis was complicated by the fact that in heart, lysophospholipase has an observed Vmax 3.6- to 4.5-fold greater than that of phospholipase A in the various subcellular fractions. Equations were derived to obtain corrected values for the activity of phospholipases A1 and A2. Using this method we found that the cytosolic and lysosomal fractions contained phospholipase A1, while the mitochondrial fractions contained primarily phospholipase A2. In heart microsomes, the positional specificity of phospholipase A could not be determined because lysophospholipase activity was very high and lysophosphatidylcholine did not accumulate.     

Phospholipase C purification and specificity with respect to individual phospholipids and brain microsomal membrane phospholipids

Phospholipase C was purified from a crude preparation derived from Cl. perfringens utilizing a one-step polypreparative electrophoresis procedure. The purified enzyme has a molecular weight of 46,500 ± 500 and is essentially free of proteolytic and phospholipase A enzymatic activities. It exhibited the following substrate specificity: PC ≥ SM > PS > PI, lyso PC. PE was hydrolyzed when PC was present.Treatment of brain microsomes with purified phospholipase C reduced membrane phospholipids by 69%. All phospholipids were attacked including PE. PC was reduced to 4% and all other phospholipids to 23–43% of their control levels. Total fatty acid composition of brain microsomes was not affected by phospholipase C action.     

Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.     

Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization

We have studied the phospholipase A2 activity in fractionated human neutrophils, employing labeled phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine as exogenous substrates. We used these phospholipid substrates labeled in the sn-1 position and measured the resulting labeled lysophospholipid forms in order to ascertain the phospholipase A2 specificity. In postnuclear supernatants from resting and A23187-activated cells, the phospholipase A2 activity showed a similar pH dependence curve with two pH optima at 5.5 and 7.5. Extracts from activated cells showed a 3-6-fold increase in enzyme activity. The subcellular distribution of phospholipase A2 activity in resting and A23187-treated human neutrophils was investigated by fractionation of postnuclear supernatants on continuous sucrose gradients. The neutral phospholipase A2 behaved as a membrane-bound enzyme and was mainly localized in the plasma membrane, the azurophilic granule, and in an ill-defined region of the gradient between the specific granules and mitochondria. The phospholipase A2 located in this undefined region showed a higher degree of activation than that located in other subcellular particulates in A23187-treated cells. This specific activation of an intracellular phospholipase A2 activity during cell stimulation indicates that cell compartmentalization may play a role in the formation of cell-activating and/or signal-transducing agents through the generation of arachidonate metabolites. Phosphatidylinositol was a better substrate for the plasma membrane enzyme, whereas phosphatidylcholine and phosphatidylethanolamine behaved as better substrates for intracellular organelle phospholipase A2 activities. The phospholipase A2 with maximal activity at pH 5.5 behaved as a soluble enzyme, and was almost completely localized in the azurophilic granules. Upon cell activation this acid enzyme activity was released in a similar way to beta-glucuronidase, a marker of azurophilic granules. These results demonstrate the different molecular properties of the phospholipase A2 activity, on the basis of its cellular location.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum.

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 X g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with an apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similarly, the free receptor also showed higher sedimentation profile with an apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI. U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the preformed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Structural studies of Fc receptors. V. Effect of phospholipase C treatment on the binding activities of the Fc receptor of macrophage or its isolated plasma membrane

The effect of phospholipase C treatment on the binding activity of the Fc receptor of guinea pig macrophage was studied to analyze the interaction of the Fc receptor with membrane phospholipids necessary for the activity. It was confirmed by subcellular fractionation that the receptor is localized on the plasma membrane. Treatment of the whole cell or isolated plasma membrane with phospholipase C of Clostridium perfringens diminished the binding of soluble IgG2-immune complex to Fc receptors on the cell or membrane. On the other hand, phospholipase C of Bacillus cereus did not affect the activity when it acted on the whole cell but it did diminish the activity when it acted on the isolated plasma membrane. Analysis of the phospholipids of untreated and treated macrophages or plasma membrane showed that phosphatidylcholine molecules, particularly those located in the membrane (not accessible to attack from the cell surface by phospholipase C of B. cereus), appear to be crucial for efficient interaction of macrophage Fc receptors with immune complex. Ligand-binding experiments with macrophages showed that the diminished binding activity was due to a decrease of the avidity for immune complex, but did not seem to be due to a decrease in the number or affinity of Fc receptors for monomeric IgG2. Taken together with the previous results which demonstrated that Fc receptors which had apparently lost the activity due to delipidation could be reconstituted with phosphatidylcholine but not with most other phospholipids, the results seem to indicate that the diminution of the binding activity to the immune complex of macrophage or its plasma membrane caused by phospholipase C treatment is due to the impairment of multivalent interaction between Fc receptor molecules on the membrane and IgG2 molecules in the immune complex, probably as a result of the loss of interaction of the head groups of phospholipids with Fc receptor molecules and the change in membrane properties resulting from the increase of diglycerides.     

Effect of membrane sterol content on the susceptibility of phospholipids to phospholipase A2

The effects of membrane sterol level on the susceptibility of LM cell plasma membranes to exogenous phospholipases A2 has been investigated. Isolated plasma membranes, containing normal or decreased sterol content, were prepared from mutant LM cell sterol auxotrophs. beta-Bungarotoxin-catalyzed hydrolysis of both endogenous phospholipids and phospholipids introduced into the membranes with beef liver phospholipid exchange proteins was monitored. In both cases, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were degraded at similar rates in normal membranes, while PC hydrolysis was specifically accelerated in sterol-depleted membranes. Additional data suggest that this preferential hydrolysis of PC is not a consequence of the phospholipid head group specificity of the phospholipase, nor of a difference in the accessibility of PC versus PE to the enzyme. Analysis of the reaction products formed during treatment of isolated membranes with phospholipase A2 showed almost no accumulation of lysophospholipids. This was found to be due to highly active lysophospholipase(s), present in LM cell plasma membranes, acting on the lysophospholipids formed by phospholipase A2 action. A soluble phospholipase A2 was partially purified from LM cells and found to behave as beta-bungarotoxin with regard to membrane sterol content. These results demonstrate that the nature of phospholipid hydrolysis, catalyzed by phospholipase A2, can be significantly affected by membrane lipid composition.     

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.     

Isolation of rat intestinal microsomes: partial characterization of mucosal cytochrome P-450

A procedure is presented for the isolation of subcellular fractions from small intestinal mucosal cells in the rat. The mucosal cells were detached by a scraping procedure resulting in an almost complete harvest of all types of cells as judged by light microscopy. Homogenization using a Potter-Elvehjem Teflon-glass device at high speed with ensuing sonication was found to be necessary for complete disruption of the cells. The subcellular fractions obtained after differential centrifugation--10,000g pellet, 105,000g pellet (microsomal fraction), and supernatant--were characterized with respect to different marker enzymes. The highest yield of 7-ethoxyresorufin-O-deethylase and NADPH-cytochrome c reductase activity in the microsomal fraction was achieved after resuspension and recentrifugation of the 10,000g pellet. Addition of anti-P-450 beta-naphthoflavone (BNF)-B2 antibodies to the incubation mixture resulted in almost complete inhibition of the O-deethylation of 7-ethoxyresorufin whereas addition of anti-P-450 phenobarbital (PB)-B2 had no effect. The presence of BNF-inducible isozymes was demonstrated by the Western blotting technique not only in intestinal microsomes from BNF-treated rats, but also in microsomes from untreated rats. Anti-P-450 BNF-B2 was also used in the peroxidase-antiperoxidase method for studies on the localization of cytochrome P-450. No BNF-inducible cytochrome P-450 could be detected in untreated rats, whereas BNF treatment resulted in a general staining of the whole villus.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with ¹²⁵I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of ¹²⁵I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed ¹²⁵I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Purification and characterization of a phospholipase A2 associated with rabbit lung microsomes: some evidence for its mitochondrial origin

Phospholipase A2 (EC 3.1.1.4) activity appeared to be unevenly distributed among the subcellular fractions of rabbit lung homogenates. The mitochondrial/lysosomal fraction, which possessed the highest specific activity, was the second most abundant source of enzyme, following the 1000 x g pellet. Crude microsomes, which were the poorest source of enzyme, had a specific activity intermediate between that of crude mitochondria and of cytosol. Despite these observations, in view of the putative role of microsomal phospholipase A2 in remodelling phosphatidylcholines for pulmonary surfactant biosynthesis, the purification of phospholipase A2 from microsomal membranes was investigated. The activity was solubilized from rabbit lung microsomes with 1 M KCl and resolved into two distinct peaks by ion-exchange chromatography. The larger peak (95% of the recovered activity) was subjected to a combination of hydroxyapatite and gel-filtration chromatography, resulting in a purification factor in excess of 70,000 relative to the microsomal membranes. There was no indication for the removal of endogenous inhibitor(s) during the purification. Application of the same purification protocol to a 1 M KCl extract of lung mitochondria resulted in phospholipase A2 profiles in each of the four columns employed that had exactly the same elution characteristics as those generated by the microsomal extracts. The purified enzyme is specific for the sn-2 ester bond of phosphatidylcholine, requires Ca2+ for activity and has an alkaline pH optimum. It is heat-labile and susceptible to treatment by p-bromophenacyl bromide and by 2-mercaptoethanol but remains unaffected by NaF, diisopropylfluorophosphate and thiol reagents.     

Prostaglandin profile and synthetic capacity of the colon: comparison of tissue sources and subcellular fractions.

Although there has been intense interest in the physiology and pathophysiology of prostaglandins (PGs) synthesized in the colon, little is known about the PG profile and synthetic capacity of different tissue sources and subcellular fractions as enzyme sources. Subcellular fractions prepared from the mucosa and muscle layer of rat colon were incubated with or without exogenous arachidonic acid ([3H]20:4n-6) for 30 min. In experiments with exogenous [3H]20:4n-6, the prostaglandin synthetic capacity of the colonic muscle layer was significantly higher than that of the mucosa. Among the subcellular fractions, microsomes had the highest PG synthetic capacity in both mucosa and muscle. The major PG product was PGI2 and PGD2 in the mucosal microsomes and PGI2 and PGE2 in the muscularis microsomes. However, production of PGI2 in the mucosa and PGE2 in the muscle was significantly reduced in the fractions containing both cytosol and microsome, resulting in an alteration of the PG profile. Substrate availability (exogenous vs endogenous supply) appears to influence the PG profile of the colon. In the colonic mucosa with exogenous [3H]20:4n-6, the production of PGI2 was 5 times higher than that of PGE2, whereas the production of PGE2 was twice higher than that of PGI2 in experiments with endogenous 20:4n-6. These observations indicate: 1) different PG profile and synthetic capacity of tissue sources and subcellular fractions; 2) alteration of PG profile due to the variation of 20:4n-6 availability. Thus, the outcome of experiments on the physiological role of PG in the colon may be determined, in part, by the tissue source and subcellular fraction selected for analysis. The present study also suggests that the variation of substrate availability in physiological and pathophysiological processes may affect the PG profile of the colon.     

Receptor-mediated endocytosis of enterokinase by rat liver. Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30-45 kDa range.     

A novel phospholipase A2 associated with nuclear matrix: stimulation of the activity and modulation of the Ca2+ dependency by polyphosphoinositides

Neutral phospholipase A2 activity, which hydrolyzed phosphatidylcholine and phosphatidylethanolamine with the same efficiency, was identified in the nuclear matrix prepared from purified nuclei of rat ascites hepatoma cells (AH 7974). The enzyme activity was optimal at pH 7.0 and required Ca2+ absolutely. Concentrations of Ca2+ for a maximal and a half-maximal activation were 1.10(-2) and 1.10(-3) M, respectively, and little activity was detected at Ca2+ concentrations lower than 1.10(-5) M. Addition of acidic phospholipids markedly stimulated the enzyme activity, and further, lowered the minimum Ca2+ concentration required for activation. In particular, the polyphosphoionositides phosphatidylinositol 4-monophosphate and 4,5-diphosphate were most effective. These two polyphosphoinositides lowered the Ca2+ concentration required for half-maximal activation to 10(-5) M and dramatically stimulated the activity at that Ca2+ concentration (greater than 30-fold). The neutral phospholipase A2 activity such as characterized in the present study was very low in the other subcellular fractions including mitochondria, microsome, plasma membrane and cytosol.     

Receptor-mediated endocytosis of enterokinase by rat liver: Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30–45 kDa range.