Localization of phosphatidylinositol 4-kinase isoenzymes in rat liver plasma membrane domains.

The presence of different isoenzymes of phosphatidylinositol 4-kinase in isolated rat liver plasma membranes and their further distribution in plasma membrane domains was examined. Both wortmannin-sensitive and -insensitive PtdIns 4-kinase activities were detected in highly purified plasma membranes obtained by aqueous two-phase affinity partitioning. The wortmannin-sensitive enzyme was identified as the 230 kDa isoform by Western blotting, whereas the 92 kDa isoform was not detected in plasma membranes. The apparent molecular weights of these isoforms were 205 and 105 kDa on SDS polyacrylamide gel electrophoresis, but approximately 500 and 230 kDa respectively on gel filtration, suggesting that both enzymes either are dimers or composed of heterologous subunits. Approximately 25% of the total 230 kDa isoenzyme present in liver, and only ca 5% of the wortmannin-insensitive one, was associated with the plasma membrane fraction. Plasma membrane domains were isolated by a combination of sucrose and Nycodenz gradient centrifugations. The 230 kDa isoform was identified in the blood sinusoidal domain, but not in the bile canalicular one, and was also found in lateral plasma membranes. The wortmannin-insensitive isoenzyme was present only in this latter material. The functional implications of this distribution of PtdIns 4-kinase isoenzymes in plasma membrane regions are discussed.

Affinity partitioning of biotinylated mixed liposomes: effect of charge on biotin--NeutrAvidin interaction.

The partitioning behaviour of biotinylated mixed liposomes in aqueous poly(ethylene glycol)/dextran two-phase systems containing NeutrAvidin-dextran suggests that the biotin-NeutrAvidin affinity interaction is charge dependent. Biotinylated phosphatidylcholine liposomes with a low negative surface charge distributed in the NeutrAvidin-containing bottom phase at neutral pH, but the introduction of additional negative charges by including phosphatidylserine or the surfactant sodium dodecylsulfate in the liposomes caused them to distribute in the poly(ethylene glycol)-rich top phase instead. By gradually lowering the pH of the affinity two-phase system below the isoelectric point (6.3) of NeutrAvidin, negatively charged phosphatidylserine/phosphatidylcholine liposomes increasingly were attracted by NeutrAvidin to the bottom phase. It is suggested that acidic amino acids present at the rim of the biotin-binding pocket of NeutrAvidin may interact electrostatically with charged residues of the closely apposed liposome surface affecting the affinity interaction.

Purification of rabbit lacrimal gland plasma membranes by aqueous two-phase affinity partitioning.

We describe the purification of lacrimal gland plasma membranes by affinity partitioning using a two-phase system containing polyethylene glycol and dextran in which wheat germ agglutinin conjugated to dextran is used as affinity ligand. When partitioning a microsomal fraction, the plasma membrane marker 5'-nucleotidase was obtained in the affinity ligand-containing bottom phase, whereas the endoplasmic reticulum marker NADH-ferricyanide reductase remained in the top phase. The affinity partitioning behaviour of components involved in exocytosis and cellular signalling was also examined.

Phosphatidylinositol 4-kinase associated with spinach plasma membranes. Isolation and characterization of two distinct forms

Highly purified plasma membranes from spinach (Spinacia oleracea L.) leaves contained phosphatidylinositol (PtdIns) kinase activity that was firmly associated with the membrane. The enzyme was solubilized by detergent treatment (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharose chromatography. Two enzymically active fractions, QI and QII, both exhibiting PtdIns 4-kinase activity, were resolved and purified 100- to 300-fold over the plasma membrane. QI and QII shared similar high apparent K(m) values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were insensitive to inhibition by adenosine. While Mg(2+) was the preferred divalent cation, Mn(2+) could partly substitute in the reaction catalyzed by the QII enzyme but not in that catalyzed by QI. Mn(2+) acted synergistically with suboptimal Mg(2+) concentrations to activate not only the QII enzyme, but also to some extent QI. Both enzymes were inhibited by millimolar concentrations of Ca(2+) and micromolar concentrations of wortmannin. The apparent molecular mass for QI was 120 kD, which was determined by SDS-PAGE and western blotting using an antibody against a peptide unique for lipid kinases and the binding of (3)H-wortmannin, and for QII 65 kD as determined by immunodetection and renaturation of PtdIns kinase activity in the 65-kD region of polyacrylamide gels.

Aqueous two-phase affinity partitioning of biotinylated liposomes using neutral avidin as affinity ligand.

Biotinylated small unilamellar liposomes were affinity partitioned in an aqueous poly(ethylene glycol)-dextran two-phase system using avidin coupled to dextran as affinity ligand. In the absence of affinity ligand more than 90% of the liposomes partitioned in the poly(ethylene glycol)-rich top phase, whereas in its presence more than 95% partitioned in the dextran-rich bottom phase. For this redistribution to occur 10 mM and above of lithium sulphate, or other appropriate salts, had to be added to the two-phase system. Without added salt the liposomes with complexed avidin-dextran instead partitioned in the top phase. An extended mixing time for the system was required for maximum redistribution. Less than two biotin residues per liposome, coupled via a C6 spacer arm, was required to redistribute the liposomes to the bottom phase.

The purification of membranes by affinity partitioning.

We describe affinity partitioning as a preparative method for membranes and membraneous structures such as organelles, cells, and viruses. Biospecific affinity partitioning is carried out in aqueous polymer two-phase systems, commonly with polyethylene glycol and dextran as phase polymers, in an environment compatible with membrane structures. Ideally, two-phase conditions are chosen to partition the bulk of membrane material into one phase, while the affinity ligand, conjugated to the second phase polymer, will selectively pull the membranes to be isolated into this phase. Suitable ligands include lectins, antibodies, and receptor-specific agents. Because the method has so far been used successfully in rather few instances, all using high ligand receptor densities in target membranes, the discussion focuses on factors to be considered when developing affinity partitioning conditions using new ligands.

Presence of a novel form of phosphatidylinositol 4-kinase in rat liver.

Rat liver microsomes contain two distinct forms of PtdIns 4-kinase which were resolved by heparin-Sepharose chromatography. One enzyme was identified as the type II PtdIns kinase previously isolated from exocytotic vesicles. The other enzyme, however, was a novel PtdIns 4-kinase isoform with properties differing from any other PtdIns kinase so far characterized. Both kinases were recognized by a monoclonal antibody specific for type II PtdIns 4-kinase, but the novel enzyme was considerably less sensitive to inhibition by adenosine and Ca2+ than type II enzymes, and in addition was specifically inhibited by submillimolar concentrations of dithioerythritol. The presence of a novel PtdIns 4-kinase isoform in rat liver raises the question of whether this enzyme is unique for this organ or whether it has a more widespread distribution but so far has avoided detection.

Phosphatidylcholine enhances the activity of rat liver type II phosphatidylinositol-kinase.

A PtdIns 4-kinase was purified extensively from rat liver exocytotic vesicles. The enzyme had a low Km for ATP, was inhibited by adenosine, and had an apparent molecular mass of 54 kDa, indicating it to be a type II PtdIns-kinase. The activity of the purified enzyme was enhanced several-fold by PtdCho, and to some extent by other phospholipids with basic polar head groups, and was inhibited by PtdSer. Kinetic analyses, presenting the substrate in mixed micelles of Triton X-100, PtdIns and PtdCho, showed that the effect of PtdCho was both to increase Vmax and to decrease the apparent Km for micellar PtdIns.

Cleavage of the alpha 1-microglobulin-bikunin precursor is localized to the Golgi apparatus of rat liver cells.

alpha 1-Microglobulin, a plasma protein with immunoregulatory properties, and bikunin, the light chain of the proteinase inhibitors inter-alpha-inhibitor and pre-alpha-inhibitor, are translated as a precursor protein from the same mRNA. The cosynthesis of alpha 1-microglobulin and bikunin is unique compared to other proproteins such as procomplement components and prohormones, since alpha 1-microglobulin and bikunin have no known functional connection. Different forms of intracellular rat liver alpha 1-microglobulin were isolated and characterized by amino acid sequence analysis, lectin binding and glycosidase treatment. Their subcellular distribution was studied by Nycodenz and sucrose gradient centrifugation, pulse-chase experiments, and electrophoresis with subsequent immunoblotting, using pro-C3 and prohaptoglobin as reference proteins. Two alpha 1-microglobulin-bikunin precursors (40 and 42 kDa), containing one and two N-linked oligosaccharides, respectively, were detected in the endoplasmic reticulum. After transport to the Golgi apparatus, the precursors were cleaved, probably C-terminal to the sequence Arg-Ala-Arg-Arg immediately preceding the bikunin part, yielding free sialylated 28 kDa alpha 1-microglobulin, representing the mature protein. The cleavage was almost complete in phosphatidylinositol 4-kinase-enriched membranes, previously identified as a post-Golgi compartment. A fourth intracellular form of alpha 1-microglobulin, 26 kDa, lacked sialic acid. None of the intracellular forms carried the yellow-brown chromophore associated with alpha 1-microglobulin when purified from serum and urine, suggesting that this chromophore becomes linked to the protein after its secretion from the liver cells.

Purification of plasma membranes by aqueous two-phase affinity partitioning.

A rapid method for purifying rat liver plasma membranes of high purity and yield is described. Squashed liver was homogenized in an aqueous polyethylene glycol-dextran two-phase system. After phase separation and reextraction of the bottom phase with fresh top phase, the combined polyethylene glycol-rich top phases were affinity partitioned in the presence of borate buffer with new bottom phase containing dextran-linked wheat-germ agglutinin. Under these conditions the lectin selectively pulled plasma membranes into the dextran-rich bottom phase, while other membranes preferentially distributed in the top phase. The lectin-containing bottom phase was reextracted with fresh top phase before collecting the purified plasma membranes by centrifugation. This protocol resulted in a preparation that was 30- to 40-fold enriched compared to the homogenate in plasma membrane markers for both the apical and basolateral domains and had yields of 55-70%. The contamination by other membranes was low. The entire procedure was completed within 90 min. The method should be useful for purifying plasma membranes also from other sources.

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.

Purification of rat liver plasma membranes by wheat-germ-agglutinin affinity partitioning.

Rat liver plasma membranes were separated from other cellular membranes by affinity partitioning in an aqueous polymer two-phase system by using the lectin wheat-germ agglutinin covalently bound to dextran as the affinity ligand. In borate buffer the bulk of membranes partitioned in the poly(ethylene glycol)-rich top phase, whereas plasma membranes were pulled selectively into the dextran-rich bottom phase in the presence of ligand. The purity and yield of plasma membranes prepared by lectin affinity partitioning and by conventional sucrose-density-gradient centrifugation was similar, as judged from marker-enzyme activities. The affinity procedure, not dependent on lengthy centrifugations, is fast and gentle and will be advantageous when studying labile components.

Purification of liver membranes highly enriched in phosphatidylinositol kinase.

Membranes highly enriched in phosphatidylinositol (PtdIns) kinase were purified from rat liver by sucrose density gradient centrifugation of a plasma membrane-depleted microsomal fraction. PtdIns kinase-containing membranes had a lower density than membranes containing Golgi and plasma membrane markers, both in sucrose and Nycodenz gradients, without being completely resolved from these other membranes. They also had a lower density than an endosomal marker. Furthermore, lectin affinity partitioning showed that PtdIns kinase did not reside in plasma membranes. PtdIns kinase in different membrane fractions was of type II and had similar kinetic properties. We suggest that the isolated membranes are the major site for phosphatidylinositol 4-phosphate formation in the liver cell, and that these membranes are part of the exocytic pathway. Thus, PtdIns kinase might be a convenient marker for the exocytic process.

Heterogeneity of smooth endoplasmic reticulum from rat liver studied by two-phase partitioning.

Smooth microsomal membranes, prepared from rat liver by sucrose-density-gradient centrifugation, were subfractionated by counter-current distribution in an aqueous two-phase system consisting of poly(ethylene glycol) and Dextran T500. A comparison of the distribution curves of marker enzymes, together with theoretically calculated curves, indicated the presence of at least five membrane subfractions, differing in the ratios of the marker enzymes. Glucose-6-phosphatase and arylesterase distributed in one manner, and NADPH-cytochrome c reductase and NADH-ferricyanide reductase in another. Evidence for further heterogeneities in the distribution of marker enzymes in smooth microsomes was obtained by analysing the membrane domain structure using a recently described method [Albertsson (1988) Q. Rev. Biophys. 21, 61-98]. Phenobarbital treatment did not influence the behaviour of the marker enzymes.

Generation of phosphatidylinositol 4,5-bisphosphate proceeds through an intracellular route in rat hepatocytes.

The subcellular distribution in rat hepatocytes of enzymes participating in the entire generation cycle of phosphatidylinositol 4,5-bisphosphate, and phosphorylated intermediates of this pathway, has been examined by Nycodenz gradient centrifugation. Our results indicate that the synthesis of phosphatidylinositol takes place in the endoplasmic reticulum, and that its phosphorylation to phosphatidylinositol 4-phosphate occurs intracellularly in low-density membranes before translocation to the plasma membrane, where it is further phosphorylated to phosphatidylinositol 4,5-bisphosphate. The intracellular formation of PIP implies a vesicular transport to the plasma membrane.

Evidence for phosphorylation of yeast phosphofructokinase.

Radioactively labelled material from yeast cells grown in the presence of [32P]phosphate was specifically recognized by antibodies raised against yeast phosphofructokinase. Purified yeast phosphofructokinase was phosphorylated in a cyclic AMP-independent manner by a protein kinase enriched from yeast extracts. This phosphorylation occurred specifically on the beta-subunit, and 0.56 mol of phosphate/mol of subunit was incorporated. The results indicate the phosphorylation of yeast phosphofructokinase both in vivo and in vitro. Phosphofructokinase phosphorylated in vitro was more stable against proteolytic degradation compared to the non-phosphorylated enzyme.

Lateral heterogeneity of rat liver plasma membranes analysed by counter-current distribution.

The lateral heterogeneity of rat liver plasma membranes was examined by fragmentation and fractionation by counter-current distribution in an aqueous two-phase polymer system. The distribution pattern was analysed by plotting the relative specific activities of marker components against each other. By this analysis asialo-orosomucoid receptors were found in a domain separated from domains containing 5'-nucleotidase and leucine aminopeptidase by another domain devoid of these markers. 5'Nucleotidase and leucine aminopeptidase resided in adjacent but separate domains. The experimental data were compared with corresponding plots of markers in model membranes. The model membranes yielded plots of different shapes depending on marker distribution and fragment size. This method of analysis should be useful for examining the lateral heterogeneity also of other membranes.

Antiproliferative response of human leukemic cells. Modulation of cytosolic protein kinase C activity by phytohemagglutinin.

Phytohemagglutinin and its isolectins PHA-E4 an PHA-L4 act antiproliferatively on an actively dividing leukemia T-cell line. Both PHA and the isolectins caused an increase in soluble protein kinase C (PK-C) activity without a corresponding decrease in particulate activity. The increase was at a maximum after 10 min and the soluble kinase activity remained high for at least 3 h. There was no direct correlation between the observed antiproliferative potency of the 2 isolectins and their ability to initially affect the distribution of PK-C activity.