Protease-activated protein kinase in rat liver plasma membrane

Upon limited proteolysis with trypsin, a cAMP and Ca2+-independent protein kinase was produced from rat liver plasma membrane. This enzyme showed a multifunctional capacity and phosphorylated calf thymus histone and rat liver ribosomal proteins. The molecular weight was estimated to be 5.0 X 10(4). When plasma membrane was treated with a buffer containing Triton X-100, a proenzyme with a molecular weight of 8.4 X 10(4) was extracted. By tryptic digestion, the proenzyme was converted to an active protein kinase which was similar to the enzyme obtained by the direct digestion of membrane. However, this proenzyme phosphorylated H1 histone in the presence of Ca2+ and phospholipid without proteolytic digestion. These results indicate the existence of a protease-activated protein kinase in rat liver plasma membrane and the proenzyme seems to be same as protein kinase C.     

Two activated forms of protease-sensitive protein kinase isolated from rat liver plasma membrane

Rat liver plasma membrane contains a protease-activated kinase which corresponds to protein kinase C. When the solubilized enzyme was digested with trypsin in the absence of NaCl, a partially activated form with an approximate molecular weight of 8 X 10(4) was produced. However, another active form of molecular weight of 5 X 10(4) was obtained when the enzyme was digested in the presence of NaCl. The larger molecular weight form was converted to the smaller form by tryptic digestion in the presence of NaCl. These results suggest the existence of two protease-activated forms of protein kinase C.     

Proteolytic activation of protein kinase C by membrane-bound protease in rat liver plasma membrane

Incubation of rat liver plasma membrane produced histone phosphorylating activity at 75 mM Mg2+ in the soluble fraction. The release of the kinase activity was inhibited by leupeptin and bovine pancreatic trypsin inhibitor, suggesting the involvement of membrane-bound protease. When partially purified protein kinase C from rat liver cytosol was treated with the trypsin-like protease purified from rat liver plasma membrane, histone phosphorylating kinase which was independent of Ca2+ and phospholipids, produced with a molecular weight of about 5 X 10(4). These results suggest that membrane-bound, trypsin-like protease activates protein kinase C in plasma membrane and the activated kinase is released from the membrane to the soluble fraction.     

Studies on protein kinase C tightly-bound to rat liver plasma membrane and its protease-activated form

1. Rat liver plasma membrane contained two types of protein kinase C which could be extracted by Ca2(+)-chelator and detergent, respectively. The activities of these two enzymes were nearly equivalent. 2. The detergent-extracted protein kinase C, tightly-bound to membrane, was separated into two subtypes by hydroxyapatite column chromatography. Based on the elution profile and the Ca2+/phospholipid requirement, the major and the minor components were identified as type III and type II protein kinase C, respectively. 3. The detergent-extracted protein kinase C was converted to an active fragment with Mr 45,000 by limited proteolysis with trypsin. Incubation under physiological level of ionic strength increased the stability of this active enzyme and protected it from further inactivation by trypsin. 4. Phosphorylation of H1 histone by the protease-activated kinase was stimulated 1.5-2-fold by phosphatidylserine. However, this enzyme phosphorylated multiple proteins in rat liver subcellular fractions in Ca2(+)- and phospholipid-independent manner. 5. These results suggest that the protein kinase C (mainly type III enzyme) tightly-bound to rat liver plasma membrane may have important role through protein phosphorylation by the native or the protease-activated kinase.     

Ionic strength-dependent interaction of rat liver casein kinase I with plasma membrane

Casein kinase I binding to rat liver plasma membrane was rapidly released from membrane by increasing the ionic strength above physiological level. The released activities at 250-300 mM NaCl were 3-4-fold higher than those obtained under lower ionic strength below 100 mM NaCl. This reaction occurred nonenzymatically and was reversible. By lowering the ionic strength from 250 mM to 50 mM NaCl by dilution at least 50% of the released enzyme was rebound to plasma membrane. By gel filtration analysis, most of the released enzyme activity under higher NaCl concentration was recovered around the molecular mass of 35,000 which corresponded to that of casein kinase I. This enzyme showed the tendency to aggregate under lower ionic strength (50 mM NaCl), but existed as monomer under higher ionic strength (250 mM NaCl). These results suggest that the release of casein kinase I from plasma membrane and the rebinding to membrane induced by the alteration of ionic strength seem to be an important regulatory mechanism in determining the subcellular distribution of this enzyme.     

Protease-activated form of protein kinase C with Mr 80,000 generated from rat liver plasma membrane by trypsin-like protease

New type of protease-activated form of protein kinase C was generated from rat liver plasma membrane by action of endogenous trypsin-like protease. The molecular mass was estimated to be about 80,000 by immunoblot analysis which was slightly smaller (approximately 2,000) than that of native protein kinase C. The protein kinase activity was 2-times stimulated by Ca2+ and phospholipid and inhibited by the synthetic peptide derived from the pseudosubstrate region of protein kinase C. This type of activated kinase was produced in purified enzyme system in the absence of either Ca2+ or phospholipid or both. These results suggest that limited proteolysis generating the active form of Mr 80,000 may occur on the inactive form of protein kinase C.     

Purification of rat liver nuclear protein kinase NI.

Rat liver nuclear protein kinase NI, which appears in the flowthrough of DEAE-Sephadex columns, has been purified approximately 15,000-fold from soluble nuclear protein with yields of up to 10%. The method of purification involved chromatography of the DEAE-flowthrough protein successively on phosvitin-Sepharose and casein-Sepharose followed by rechromatography on phosvitin-Sepharose. The purified enzyme has an s20,w and molecular weight of 3.7 and 47,000, respectively, as determined by sucrose density gradient centrifugation in 0.4 M NaCl. A similar molecular weight of 42,000 was determined by gel filtration using Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide with a molecular weight of 25,000. Protein kinase NI therefore consists of a dimer of two identical subunits. Protein kinase NI exhibits maximal activity on casein substrate and is not stimulated by 10(-5) to 10(-4) M cAMP or cGMP when either casein or histone H2b is used as a substrate.     

Characterization of glycyrrhizin-binding protein kinase from the crude membrane fraction of rat liver

Using GL-affinity column chromatography, a casein phosphorylating protein kinase was purified selectively from the crude membrane fraction of rat liver. The biochemical characteristics of the purified kinase (approximately Mr 210 kDa) are very similar to those reported for polypeptide-dependent protein kinase (kinase P). Moreover, low doses of GL selectively inhibit phosphorylation of Mr 35-36 kDa polypeptides (which are cross-reacted with anti-lipocortins I and II) by the kinase in vitro. These results suggest that the anti-inflammatory activity of GL may involve the impairment of the physiological functions of lipocortins through their specific modification by the kinase at the cell membrane level.     

Effect of a sunflower oil-supplemented diet on protein kinase activities of rat liver plasma membranes.

1. The effect of a sunflower oil-enriched diet on plasma membrane-bound protein kinase C, protein kinase A, casein and tyrosine kinase activities was studied. 2. The diet induced an increase in the content of linoleic acid and a decrease in the content of palmitic acid. The anisotropy parameter (rs) of the fluorescence probe DPH and SDPH decreased strongly in the experimental group. 3. Protein kinase C was stimulated more than two times. Tyrosine kinase, protein kinase A and casein kinase activities were increased by 65, 57 and 40%, respectively. 4. We suggest that a more fluid lipid environment favours higher plasma membrane-bound protein kinase activities.     

Cholesteryl ester hydrolytic acitivity of rat liver plasma membrane.

Cholesteryl ester hydrolyzing activity of rat liver plasma membranes was studied using acetone-dispersed [4-14-C] cholesteryl oleate as substrate. In contrast to whole liver homogenates which displayed ample activity at both acid (4.5) and neutral (6.2-7.4) pH, purified plasma membrane fractions contained little activity at neutral pH as compared to acid pH. Moreover, rate-zonal sucrose density-gradient centrifugation patterns of plasma membrane rich fractions suggested a specific association with plasma membrane only in the case of the acid activity. These findings suggest that in vivo hepatic cell surface membranes contain little or no cholesteryl ester hydrolytic activity at extracellular pH. They support the possibility that plasma lipoprotein cholesteryl esters enter hepatic parenchymal cells prior to hydrolysis.     

Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-galactosamine.

The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.     

Biogenesis of plasma membrane glycoproteins. Purification and properties of two rat liver plasma membrane glycoproteins

As a preliminary to a study of the biogenesis of individual plasma membrane glycoproteins, the marker enzyme nucleotide pyrophosphatase (NPPase) and a major rat liver plasma membrane sialoprotein, subsequently found to be identical with the enzyme dipeptidyl peptidase IV (DPP IV), were purified 10,000- and 2,000-fold, respectively, from rat liver. Both were amphipathic proteins which formed defined micellar complexes with detergents and aggregated in their absence. Gel filtration, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the Triton X-100 complex of NPPase to contain a single 150,000-dalton peptide, while that of DPP IV was composed of two 120,000-dalton subunits; each complex also contained about 150,000-dalton Triton X-100. Trypsin cleaved the detergent complexes with release of major hydrophilic fragments which no longer bound detergent micelles; the accompanying change in peptide size was small for NPPase and undetectable for DPP IV, which also retained the dimer structure of its native form. DPP IV was the only major glycoprotein in rat liver plasma membrane which bound strongly to wheat germ agglutinin. Monospecific rabbit antibodies against NPPase and DPP IV precipitated the antigens without affecting their enzymatic activities.     

Effect of triiodothyronine on rat liver chromatin protein kinase.

1. Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2. Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32P when incubated with [gamma-32P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins.     

Hemoglobin-haptoglobin receptor in rat liver plasma membrane

The presence of a receptor specific for the hemoglobin . haptoglobin complex is demonstrated in rat liver plasma membranes. Hemoglobin . haptoglobin complex, administered intravenously to rats, was cleared from the circulation at a constant rate with exclusive incorporation of the molecule into hepatocytes. This incorporation was unaffected by the simultaneous injection of asialoglycoprotein or heme . hemopexin complex. In vitro experiments with isolated liver plasma membranes indicated the absence of competitive binding of these molecules to the membrane and suggested that this receptor might recognize an altered conformation of the haptoglobin moiety of the complex resulting from the binding with hemoglobin. These observations suggest that the mechanism of recognition and binding of hemoglobin . haptoglobin complex by the receptor is different from that of the asialoglycoprotein receptor or heme . hemopexin receptor.     

Identification of a detergent-solubilized plasma membrane protein from rat liver recognizing apolipoprotein A-IV

Apolipoprotein A-IV (apo A-IV) is present in plasma associated to both HDL and as a complex with lipids that cannot be floated by ultracentrifugation at 1.21 g/ml density. Apo A-IV is likely an important molecular determinant in HDL binding to the liver. In this communication, data are presented supporting the view that a specific liver plasma membrane protein of Mr 95,000 is a constituent of the apo A-IV binding site. The protein was solubilized with CHAPS from purified rat liver plasma membranes and subjected to SDS-PAGE. Transblotted to nitrocellulose sheet could be identified as recognizing 125I-apo A-IV-DMPC by autoradiography. 125I-apo A-I-DMPC and radioiodinated rat apo E-poor HDL, also bound to the protein. Apo B-100 (as human LDL) and apo C-III did not bind. The protein identified is likely to be the same that has been previously identified by Graham and Oram [1987) J. Biol. Chem. 262, 7439-7442) as 'HDL receptor protein'.     

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-activated ATPase of rat liver plasma membrane.

Rat liver plasma membranes hydrolyze ATP in the presence of Ca2+. The rate of hydrolysis is different when Mg2+ions are present in the incubation system. Several parameters differentiate Ca2+-ATPase from Mg2+-ATPase: a) the Km of ATP hydrolysis for Ca2+ (2.25 x 10(-4) M) is lower than for Mg2+ (2.14 x 10(-3) M); b) the shape of the activation curve is hyperbolic in the presence of Ca2+ and sigmoid in the presence of Mg2+; c) Mg2+-ATPase shows two different values of activation energy while Ca2+-ATPase presents only a single value; d) Ca2+-ATPase is inhibited, while Mg2+-ATPase is unaffected by cyclic AMP. Ca2+-ATPase is localized on the plasma membrane and is not inhibited by cysteine. It does not hydrolyze substrates different from nucleotides triphosphate, such as glucose-1-phosphate or alpha-glycero-phosphate. The enzyme is probably related to a mechanism of calcium transport.     

Cyclic nucleotide-independent protein kinase from rat liver. Purification and characterization of a multifunctional protein kinase

A rat liver cAMP-independent protein kinase that phosphorylates peptide b of ATP-citrate lyase (Ramakrishna, S., Pucci, D. L., and Benjamin, W. B. (1983) J. Biol. Chem. 258, 4950-4956) has been purified to apparent homogeneity. The molecular weight, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sucrose density gradient, and by gel filtration, was found to be 36,000. This protein kinase phosphorylates in vitro ATP-citrate lyase, acetyl-CoA carboxylase, and glycogen synthase and does not phosphorylate phosphorylase, phosphorylase kinase, histone, phosvitin, and casein. It has Fa (activity factor) activity stimulating the ATP X Mg-dependent phosphatase and is therefore named a multifunctional protein kinase. This kinase differs from glycogen synthase kinase-3 with regard to substrate specificity, kinetic parameters, and physicochemical properties.