Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria.

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80-90% was found in the intermembrane fraction, while the rest was associated with mitoplasts. The intermembrane protein kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplasts. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinases, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.

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.

Determination of Ca2+- and phospholipid-dependent protein kinase in rat liver membranes.

A method has been developed to measure the Ca2+- and phospholipid-dependent protein kinase in membrane fractions. The method is based on the fact that this enzyme is resistant to comparatively high concentrations of octylglycoside. Rat liver membranes were treated with octylglycoside and the phosphate incorporation from [gamma-32P]ATP was measured in the presence of histone H1. The enzyme activity was determined as the difference between the incorporation obtained after addition of Ca2+ and phosphatidylserine and the incorporation obtained without these additions but with EGTA. The endogenous incorporation of phosphate to membrane components was constant under these incubation conditions. The conditions for determination of the membrane-bound enzyme were optimized. Two thirds of the total enzymic activity was attached to membranes in rat liver cells. A highly purified plasma membrane preparation had the highest specific activity, while most of the bound enzyme was found in microsomes, and only traces were found in mitochondria.

Activation of phosphatidylinositol-4-phosphate kinase in rat liver plasma membranes by polyamines.

The formation of phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous substrate in rat liver plasma membranes was stimulated approximately 3-fold by 1 mM spermine, with half-maximal effect at 0.2 mM polyamine. This effect of spermine was due to enhancement of phosphatidylinositol-4-phosphate kinase activity rather than to a decrease in degradation of PIP2 formed or the substrate phosphatidylinositol 4-phosphate (PIP). The stimulation of phosphatidylinositol-4-phosphate kinase by spermine decreased to half at physiological ionic strength, and was not affected appreciably by variations in the concentration of ATP and MgCl2. Among several di- and polyamines only spermine and spermidine were effective. Although spermine may cause aggregation of membrane vesicles, thereby potentially increasing substrate availability for phosphatidylinositol-4-phosphate kinase, our results do not support such an explanation for the enhancement in enzyme activity. Phosphatidylinositol kinase activity, contrary to phosphatidylinositol-4-phosphate kinase, was not stimulated appreciably by spermine.

Removal of nonionic detergent from proteins fractionated by electrofocusing.

The nonionic detergent Nonipol TD 12 (an alkyl polyoxyethylene alcohol) has been removed from solubilised proteins after their fractionation by electrofocusing. Following electrofocusing in nonionic detergent an anionic or cationic detergent was added to the focussing medium and the focusing was allowed to continue. The ionic detergent formed mixed micelles with the nonionic detergent. Thus charged, the mixed micelles migrated to either electrode, removing nonionic detergent from the fractionated proteins. Applying this technique to an adipose tissue preparation, detergent-inhibited activity of a lipolytic enzyme was restored and the binding of adenosine 3':5'-cyclic monophosphate (cyclic AMP) to a protein kinase was increased.

Subcellular localization and enzymatic properties of rat liver phosphatidylinositol-4-phosphate kinase.

The phosphatidylinositol-4-phosphate kinase activity in rat liver showed a subcellular distribution different from that of phosphatidylinositol kinase. It was preferentially associated with plasma membrane-rich subcellular fractions, while no or minimal activity could be ascribed to mitochondria, lysosomes, Golgi membranes or the endoplasmic reticulum. The plasma membrane enzyme phosphorylated endogenous and exogenously added phosphatidylinositol 4-phosphate at comparable initial rates. The phosphorylation of endogenous substrate was strongly inhibited by Triton X-100, while the phosphorylation of added substrate was enhanced, suggesting that endogenous phosphatidylinositol 4-phosphate was readily available to the enzyme in unperturbed plasma membranes. The total activity of phosphatidylinositol-4-phosphate kinase in rat liver was only 1/20 that of phosphatidylinositol kinase. The enzyme activity showed an unusually broad pH-optimum in the neutral range. Mg2+ was the preferred divalent cation and Km towards ATP was about 3-fold higher than the corresponding value for phosphatidylinositol kinase.

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.

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.

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.

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.

Microsomal Ca2+- and phospholipid-dependent protein kinase. Identification and in vitro binding studies.

A Ca2+- and phospholipid-dependent protein kinase (CaPK) has been identified in rat liver microsomes. CaPK isolated from liver cytosol bound to smooth microsomes in the presence of 100 microM CaCl2. A saturation in binding was observed when a 5-fold excess of enzyme over that present in microsomes had become bound. The microsomal CaPK and 50% of the enzyme bound in vitro was not removed by EGTA treatment. This suggests that Ca2+ is required for the binding of CaPK to microsomes, but not for the retention of the enzyme on the membrane.

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.

Phospholipase C in rat liver plasma membranes. Phosphoinositide specificity and regulation by guanine nucleotides and calcium.

Phospholipase C activity against phosphoinositides in isolated rat liver plasma membranes has been examined using exogenous substrates. The enzyme hydrolyzed phosphatidylinositol 4,5-bisphosphate 30-40-times faster than phosphatidylinositol 4-monophosphate, while phosphatidylinositol was not a substrate. Maximum activity was observed with 1.1 mM phosphatidylinositol 4,5-bisphosphate at pH 5.0. The enzyme was stimulated by micromolar concentrations of Ca2+. The GTP analogue guanylyl (beta,gamma-methylene)diphosphonate enhanced phospholipase C activity at and above 0.3 microM Ca2+, but was inhibitory at 0.1 microM Ca2+. This supports the suggestion that plasma membrane phospholipase C is regulated by guanine nucleotide-binding protein, but indicates a regulatory mechanism different from that of other enzymes regulated by such proteins.

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.

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.

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.

Identification of Ca2+-stimulated polyphosphoinositide phospholipase C in isolated plant plasma membranes.

A polyphosphoinositide phospholipase C has been identified in highly purified plasma membranes from shoots and roots of wheat seedlings. The enzyme preferentially hydrolysed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate and had a different phosphoinositide substrate profile from soluble phospholipase C. The enzyme activity was lower in plasma membranes isolated from light-grown shoots than from dark-grown ones, whereas no differences in activity between plasma membranes from light- and dark-grown roots were seen. Maximum activity of the membrane-bound enzyme was observed around pH 6. It was activated by micromolar concentrations of Ca2+, but not by GTP or GTP analogues. The enzyme may participate in signal transduction over the plant plasma membrane.

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.

Enzyme immunoassay of benzo[a]pyrene conjugated to DNA, RNA and microsomal proteins using a monoclonal antibody.

An enzyme immunoassay for the detection of benzo[a]pyrene covalently conjugated to macromolecules has been developed. The monoclonal antibody, raised through in vitro immunization reacted with benzo[a]pyrene metabolites bound to DNA, RNA and proteins. The lower detection limit for the assay was 1 pmol for benzo[a]pyrene bound to DNA or RNA, and 5 pmol when bound to protein.

Covalent binding of benzo[a]pyrene to rat liver cytosolic proteins and its effect on the binding to microsomal proteins.

Benzo[a]pyrene will bind covalently to rat liver cytosolic proteins when incubated with microsomes and NADPH. The binding is most extensive when microsomes from 3-methylcholanthrene-treated rather than phenobarbital-treated or control rats are used. The binding to cytosolic proteins increases when incubations are performed with increasing concentrations of cytosol. At the same time the covalent binding of benzo[a]pyrene to microsomal proteins decreases. Two cytosolic polypeptides are the main targets for benzo[a]pyrene. These have the same mobility in polyacrylamide gels as the subunits of purified glutathione S-transferase B. These subunits also react covalently with benzo[a]pyrene when the transferase is incubated with microsomes and NADPH.