Secretory PAF-acetylhydrolase of the rat hepatobiliary system: characterization and partial purification.

Hepatocytes and Kupffer cells in primary culture both secrete plasma-type platelet-activating factor-acetylhydrolase (pPAF-AH) into serum-free culture medium. The rate of secretion of pPAF-AH by Kupffer cells was 20 to 25 times higher than from hepatocytes, and Kupffer cells expressed a higher level of pPAF-AH mRNA than did hepatocytes. Purified liver cell-secreted pPAF-AH exhibited a major protein band of 65-67 kDa on SDS-PAGE; this was the band predominantly labeled when the enzyme catalytic center was reacted with [3H]diisopropylfluorophosphate ([3H]DFP). Rat bile collected from cannulated bile ducts contained significant PAF-AH activity, and bile samples possessed a prominent band at 30-32 kDa, which was the exclusive target for [3H]DFP. Experiments using tunicamycin, an inhibitor of N-linked glycosylation, and endoglycosidase H suggested that pPAF-AH secreted constitutively by cultured hepatocytes and Kupffer cells is glycosylated. The present study supports the notion that hepatic secretion of pPAF-AH into the blood contributes to the regulation of PAF and oxidized phospholipid levels in the circulation, whereas secretion of PAF-AH into the bile may allow hepatic control of these phospholipid signaling molecules in the gastrointestinal tract.

Dissociation and reassembly of the dihydrolipoyl transacetylase component of the bovine heart pyruvate dehydrogenase complex.

The catalytic activity and the state of aggregation of the dihydrolipoyl transacetylase-lipoamide dehydrogenase binding protein (E2-E3BP) subcomplex of the bovine heart pyruvate dehydrogenase multienzyme complex were investigated. Treatment of E2-E3BP with the chaotropic salts GndnCl or KSCN led to a rapid decrease in transacetylase activity which was accompanied by a loss of the native quaternary structure, as indicated by changes in the sedimentation properties of the E2-E3BP subcomplex. Reassembly or refolding of dissociated E2-E3BP was achieved for the GndnCl-treated subcomplex using a defined protocol. This reassembly procedure effectively excluded all E3BP from the reassembled oligomeric transacetylase. The reassembled oligomeric E2, free of E3BP, was unable to reconstitute the overall activity of the complex following incubation with pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3). In binding studies using radiolabeled components it was demonstrated that the reassembled transacetylase, while retaining its capacity for reductive acetylation and its ability to bind E1, lost its ability to bind E3. The evidence presented in this study indicates that the strong association of E3BP with E2 facilitates the binding of E3, the lipoamide dehydrogenase component, and therefore may have an important role in the assembly and ultimately the catalytic activity of the pyruvate dehydrogenase multienzyme complex.

Pyruvate dehydrogenase multienzyme complex. Characterization of assembly intermediates by sedimentation velocity analysis.

The pyruvate dehydrogenase complex is a large, highly organized assembly of several different catalytic and regulatory component enzymes. The structural core of the complex is the E2-X subcomplex, consisting of 60 dihydrolipoamide transacetylase (E2) subunits arranged in a pentagonal dodecahedron; 6 protein X and 2 pyruvate dehydrogenase kinase molecules are tightly associated with this E2 60-mer. The native E2-X subcomplex exhibits a sedimentation coefficient of 32 S. The effects of several chaotropes (guanidinium chloride, potassium thiocyanide, and urea) on the E2-X subcomplex were assessed. Treatment of the E2-X subcomplex with 4 M guanidinium chloride caused a complete loss of enzymatic activity and the dissociation of the subcomplex into monomeric 1.5-3 S species. Removal of the chaotrope by dialysis for 18 h resulted in complete restoration of E2 enzymatic activity and reassembly of a 32 S subcomplex; this reassembled subcomplex contained less protein X than the native subcomplex. Sedimentation velocity analysis of reassembled E2-X subcomplex demonstrated the presence of an 8 S assembly intermediate; this sedimentation coefficient is characteristic of globular proteins of molecular weights similar to that expected for a trimer of E2. Shorter periods of dialysis also gave rise to the 8 S species; the amount of this intermediate decreased with increasing times of dialysis. The 8 S species associated non-cooperatively to yield additional assembly intermediates exhibiting sedimentation coefficients of 10-32 S.

Platelet-activating factor-mediated synthesis of prostaglandins in rat Kupffer cells.

Synthesis of prostaglandins was stimulated in rat Kupffer cells upon challenge with platelet-activating factor (PAF). PAF-mediated synthesis of prostaglandins was inhibited by the Ca2+ ion chelator (EGTA), the Ca2+ channel antagonist (nifedipine) and U66985, a structural analogue and antagonist of the biological effects of PAF in other cellular systems. Inhibitors of protein kinase C, staurosporine and polymixin B, did not affect PAF-induced prostaglandin synthesis. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, stimulated synthesis of prostaglandins in Kupffer cells; PAF and PMA exerted additive actions on this process. Both PAF- and PMA-stimulated prostaglandin production was inhibited by TMB-8. PAF-stimulated synthesis of prostaglandins also was inhibited upon treatment of Kupffer cells with pertussis toxin. Cholera toxin, in contrast, stimulated the production of prostaglandins in a concentration-dependent manner; cholera toxin and PAF together had an additive effect. These results suggest that PAF-induced synthesis of prostaglandins is stimulated via a specific receptor coupled to a pertussis toxin-sensitive G-protein, is dependent upon extracellular Ca2+ and is not influenced by protein Kinase C activation. Since PAF and prostaglandins are produced in the liver under conditions such as endotoxemia, PAF-mediated synthesis of these lipid autacoids may be of importance in the regulation of hepatic function during pathophysiological episodes.

Size determination of multienzyme complexes using two-dimensional agarose gel electrophoresis.

In studies of the size and structure of multienzyme complexes, a procedure complementary to electron microscopy for determining the molecular dimensions of hydrated multisubunit complexes is needed. For some applications this procedure must be capable of detecting aggregation of complexes and must be applicable to impure preparations. In the present study, a procedure of two-dimensional agarose gel electrophoresis (2d-AGE) (Serwer, P. et al. Anal. Biochem. 152:339-345, 1986) was modified and employed to provide accurate size measurements of several classical multienzyme complexes. To improve band clarity and to achieve required gel pore sizes, a hydroxyethylated agarose was used. The effective pore's radius (PE) as a function of gel concentration was determined for this agarose in the range of PE values needed for multienzyme complexes (effective radius, R = 10-30 nm). Appropriate conditions were established to measure R values +/- 1% of the pyruvate (PDC), alpha-ketoglutarate (alpha-KGDC), and the branched chain alpha-keto acid (BCDC) dehydrogenase multienzyme complexes; the accuracy of R was limited by the accuracy of the determinations of the R value for the size standards. The PDC from bovine heart was found to have an R = 22.4 +/- 0.2 nm following cross-linking with glutaraldehyde that was necessary for stabilization of the complex. Dimers and trimers of PDC, present in the preparations used, were separated from monomeric PDC during 2d-AGE.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of acetylglyceryl ether phosphorylcholine on glycogenolysis and phosphatidylinositol 4,5-bisphosphate metabolism in rat hepatocytes.

The effect of acetylglyceryl ether phosphorylcholine (AGEPC) on glycogenolysis and phosphatidylinositol 4,5-bisphosphate has been studied in rat hepatocytes. Previously, this laboratory demonstrated that AGEPC stimulated glucose output from the perfused rat liver and promoted the breakdown of phosphoinositides in rat hepatocytes (Shukla, S. D., Buxton, D. B., Olson, M.S., and Hanahan, D.J. (1983) J. Biol. Chem. 258, 10212-10214). In the present study, addition of AGEPC (10(-13) to 10(-9) M) to rat hepatocytes failed to stimulate glucose output, whereas epinephrine (10(-5)M) and glucagon (10(-7)M) stimulated glucose output by 100% or more in these same cells. The effects of AGEPC, epinephrine, vasopressin, and glucagon on glycogen phosphorylase activity and the breakdown of phosphatidylinositol 4,5-bisphosphate were compared in hepatocytes. AGEPC (10(-9)M) promoted the breakdown of phosphatidylinositol 4,5-bisphosphate in a fashion similar to epinephrine (10(-5)M) and vasopressin (10(-7)M). In contrast to the two calcium-mobilizing hormones, epinephrine and vasopressin, AGEPC did not cause an activation of glycogen phosphorylase. Glucagon activation of glycogen phosphorylase was not accompanied by a significant effect on phosphatidylinositol 4,5-bisphosphate hydrolysis. Thus, AGEPC is a chemical mediator which induces the degradation of phosphatidylinositol 4,5-bisphosphate without activating glycogenolysis in hepatocytes.

The analysis of acyl-coenzyme A derivatives by reverse-phase high-performance liquid chromatography.

A method for determining tissue levels of Coenzyme A and various short-chain-length acyl-CoA derivatives using high-performance liquid chromatography is presented. Separation of the various compounds was accomplished using a reverse-phase Spherisorb ODS II, 5-microns C18 column. Mobile-phase solvents were (a) potassium phosphate, 220 mM; thiodiglycol (2,2-thiodiethanol), 0.05% (v/v), pH 4.0 and (b) methanol, 98%; chloroform; 2% (v/v). The various acyl-CoA derivatives were detected by monitoring the column effluent at 254 nm. Nearly baseline separation was obtained for a standard mixture of free CoASH, methylmalonyl-CoA, beta-hydroxy-beta-methylglutaryl-CoA, succinyl-CoA, acetoacetyl-CoA, acetyl-CoA, propionyl-CoA, isobutyryl-CoA, beta-methyl-crotonyl-CoA, and isovaleryl-CoA. CoA derivative profiles were determined in neutralized perchloric acid extracts of perfused rat hearts and livers and of isolated rat liver mitochondria to demonstrate the utility of this method for assessing the levels of CoA derivatives in biological samples.

The effect of propionate on the regulation of the pyruvate dehydrogenase complex in the rat liver.

Propionate inhibited the metabolic flux through the pyruvate dehydrogenase reaction in the perfused rat liver when the perfusate concentration of propionate was below 10 mM and the perfusate pyruvate concentration was held within the physiological range. At higher propionate concentrations (e.g., 20 mM) the inhibition of pyruvate dehydrogenase was alleviated and the activation state of the pyruvate dehydrogenase complex was nearly doubled. In livers perfused with a high pyruvate concentration (e.g., 5 mM), propionate coinfusion at all concentrations inhibited the rate of pyruvate decarboxylation. Additional studies were performed in liver mitochondria maintained in State 3 where the ATP/ADP and the NADH/NAD+ ratios were held constant. Low propionate concentrations (e.g., 0.5 mM) inactivated the mitochondrial pyruvate dehydrogenase complex, whereas propionate levels in excess of 1 mM activated the enzyme complex. CoA distribution analyses of the mitochondrial incubations indicated that the presence of either 0.5 or 10 mM propionate caused a substantial accumulation of propionyl-CoA and methylmalonyl-CoA at the expense of free CoASH. Experiments were performed in which the ratios of various acyl-CoA derivatives to CoASH were varied by sequentially increasing the L-carnitine concentrations in the incubation. An inverse relationship between the propionyl-CoA/CoASH and methylmalonyl-CoA/CoASH ratios and the activity of the pyruvate dehydrogenase complex was observed. Experiments using freeze-thawed liver mitochondrial membranes indicated that propionate protected the pyruvate dehydrogenase complex from ATP-mediated inactivation by the pyruvate dehydrogenase kinase. It is our contention that the inactivation of pyruvate dehydrogenase complex at low propionate levels may be due to an increase in the mitochondrial acyl-CoA/CoASH ratios, whereas the activation of the enzyme complex demonstrated at high propionate levels is due to the inhibition of the pyruvate dehydrogenase kinase in a manner similar to that caused by pyruvate or dichloroacetic acid.

Studies on the regulation of the branched chain alpha-keto acid dehydrogenase in the perfused rat liver.

The regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex was investigated in the isolated, perfused rat liver. The metabolic flux through the branched chain alpha-keto acid dehydrogenase was monitored by measuring the production of 14CO2 from infused 1-14C-labeled branched chain alpha-keto acid substrates. The rate of decarboxylation of alpha-keto[1-14C]isocaproate exceeded that of alpha-keto[1-14C]isovalerate at all concentrations of the substrates infused. Coinfusion of either alpha-ketoisovalerate or alpha-keto-beta-methylvalerate inhibited the rate of alpha-keto[1-14C]isocaproate decarboxylation. The rate of alpha-keto[1-14C]isovalerate decarboxylation ws enhanced during coinfusion of L(--)carnitine, while alpha-keto[1-14C]isocaproate decarboxylation was unaffected. The presence of pyruvate in the perfusion medium resulted in an inhibition of the flux through the branched chain complex with either alpha-ketoisocaproate or alpha-ketoisovalerate as the substrate. DL-beta-hydroxybutyrate infusion inhibited alpha-keto[1-14C]isocaproate decarboxylation by 18% but resulted in nearly a 100% stimulation of alpha-keto[1-14C]isovalerate decarboxylation. The evidence presented indicates that (alpha) the metabolic flux through the branched chain alpha-keto acid dehydrogenase complex can be monitored effectively in a continuous fashion in the perfused liver by following the release of 14CO2 from infused 1-14C-labeled substrates and (b) the changes observed in the metabolic flux through the branched chain complex during coinfusion of alternative substrates and other compounds may be entirely different depending upon which branched chain alpha-keto acid substrate is utilized to monitor this reaction.

Studies on the activation and inactivation of the branched chain alpha-keto acid dehydrogenase in the perfused rat heart.

Evidence for a reversible process resulting in stable activated and inactivated states of the mitochondrial branched chain alpha-keto acid dehydrogenase complex in isolated perfused rat heart is presented. The inactivation process is mediated by pyruvate infusion, while activation (up to 18-fold) is facilitated by branched chain alpha-keto acid substrates. The low activity state of the branched chain complex characteristic of freshly excised rat hearts could be maintained by infusion of either pyruvate or glucose. Activation of the complex in the perfused rat heart was achieved slowly by substrate-free perfusion, while rapid activation was accomplished by infusion of branched chain alpha-keto acids. The fully activated enzyme complex resulting from branched chain alpha-keto acid infusion subsequently could be inactivated maximally by infusion of pyruvate alone or intermediate degrees of inactivation could be produced by certain ratios of co-infused pyruvate and branched chain alpha-keto acid. alpha-Ketoisocaproate was an order of magnitude more effective than alpha-keto isovalerate either in preventing inactivation or in stimulating the opposing activation process when co-infused with pyruvate. The mitochondrial pyruvate transport inhibitor, alpha-cyanocinnamate, effectively prevented inactivation of the complex by infused pyruvate. Differential changes in the activation states of the branched chain alpha-keto acid dehydrogenase and pyruvate dehydrogenase complexes were evident when the two complexes were compared in apparently similar flux-inhibited (via octanoate infusion) and flux-stimulated (via dichloroacetate infusion) metabolic conditions. The differential effect of pyruvate concentration on the activity states of the two complexes was also well-defined. The results of the present study suggest distinct systems for the regulation of the activity of the two multienzyme complexes of interest. While our results argue neither for nor against an inactivation of the branched chain alpha-keto acid dehydrogenase complex by a protein kinase, the regulatory properties of such an intramitochondrial protein kinase may not be similar to the pyruvate dehydrogenase kinase. The mechanistic nature of the suggested novel regulatory system concerned with the pyruvate-mediated inactivation of the branched chain alpha-keto acid activation cannot be inferred at the present time.

Configuration of unsheared nucleohistone. Effects of ionic strength and of histone F1 removal.

Nucleohistone solubilized from rabbit thymus nuclei by an endogenous nuclease has in 0.15 M salt an exceptionally low intrinsic viscosity and very high sedimentation velocity. A fully reversible expansion of configuration occurs on lowering ionic strength. When [eta] is plotted against I-1/2 and extrapolated to high I, [eta] = 0 is reached at I = 0.4-1 M and [eta] at I = infinity is negative, contrary to the behavior of DNA and of the great majority of polyelectrolytes, which extrapolate to a positive [eta] at I = infinity. This behavior demands that the configuration of nucleohistone depends not only on electrostatic expansive forces but also on contracting forces which are not electrostatic and do not go to zero in any accessible configuration. Intramolecular hydrophobic bonds might provide such contracting forces. Increasing I above 0.15 M leads to precipitation near 0.3 M and redissolution with dissociation of F1 and expansion in 0.6 M. The expansion is largely but not completely reversed on return to 0.15 M. Much further expansion occurs in I = 1.2 M. Nucleohistone exposed to 1.2 M could not be redissolved in the original medium. Nucleohistone depleted of F1 exhibits a similar expansion as ionic strength is reduced, at higher viscosities throughout. On extrapolation to I = infinity both positive and negative viscosities were observed, on different lots, perhaps reflecting variable extraction of other histones. Circular dichroism spectra are very little affected by ionic strength (0.6 M and lower) or F1 removal, despite tenfold changes in viscosity.