Arachidonic acid metabolim in rat pancreatic acinar cells: Calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [¹⁴C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromtography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8–10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, and PGE₂ were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Subcellular distribution of the mannan-binding protein and its endogenous inhibitors in rat liver

The subcellular distribution of the mannan-binding protein from rat liver, a lectin specific for mannose and N-acetylglucosamine, was studied. Approximately 75% of the binding activity of the homogenate was recovered in microsomes, approximately 76% of which was accounted for by rough microsomes. Rough microsomes had the highest specific activity of binding, followed by the Golgi apparatus and smooth microsomes, whereas plasma membranes, lysosomes, mitochondria, and the soluble fraction had little or no binding activity. A topographical survey indicated that the binding protein was localized exclusively on the cisternal surface of microsomal vesicles. Thus, the binding protein of microsomal vesicles was protected from protease digestion and was released from the vesicles by mild detergent treatment. Competitive inhibitors, which presumably represent endogenous ligands of the binding protein, were found among subcellular fractions. More than 50% of the inhibitory activity of the homogenate was recovered in rough microsomes, while the highest specific activity of inhibition was found in lysosomes. The K_i values estimated for rough microsomes and lysosomes were 25.9 and 8.67 μg/ml, respectively. The distribution profiles of inhibitors were correlated roughly with those of the binding protein, resulting in masking of the binding activity in organelles up to the level of 86%. On the basis of the known localization and topology of the binding protein and endogenous inhibitors (ligands), possible physiological functions of the binding protein relevant to the transport of biosynthetic intermediates of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus and from the Golgi apparatus to lysosomes were discussed.     

Purification and crystallization of rat liver fatty acid synthetase

A purification procedure for rat liver fatty acid synthetase has been developed using polyethylene glycol. This procedure results in high yields of the enzyme which is essentially free of endogenous proteolytic nicking and also free of any contaminating proteases. The fatty acid synthetase obtained has a specific activity range of 1.8–2.1 measured at 25 °C and is stable at 4 °C for a few weeks and indefinitely when frozen. Approximately 1 mg of enzyme can be obtained per gram of induced rat liver. The enzyme is pure as determined by sodium dodecyl sulfate-gel electrophoresis, sedimentation velocity, and immunoelectrophoresis. The first crystallization of rat liver fatty acid synthetase is also reported.     

Arachidonic acid, 12- and 15-hydroxyeicosatetraenoic acids, eicosapentaenoic acid, and phospholipase A2 induce starfish oocyte maturation

In starfish oocyte maturation (meiosis reinitiation) is induced by the natural hormone 1-methyladenine (1-Me-Ade). This paper shows that arachidonic acid (AA) induces oocyte maturation at concentrations above 0.5 microM. This maturation shares many characteristics with 1-MeAde-induced maturation: same kinetics, same required contact time, same stimulations of protein phosphorylation and sodium influx. Although calcium facilitates the AA-induced but not the 1-MeAde-induced maturation, AA, like 1-MeAde, does not stimulate the uptake of calcium. Calcium does not facilitate the uptake of AA by oocytes. Out of 36 different fatty acids (saturated and unsaturated), only eicosatetraenoic (AA) and eicosapentaenoic acids were found to mimic 1-MeAde. Calcium-dependent phospholipases A2 from bee venom and Naja venom also induce maturation (0.1-1 unit/ml) when added externally to the oocytes. Phospholipase A2 inhibitors (quinacrine, bromophenacylbromide) block maturation; inhibition is reversed by increasing the 1-MeAde concentration and only occurs during the hormone-dependent period. AA is usually metabolized through oxidation by cyclooxygenase or lipoxygenase. Cyclooxygenase inhibitors (acetylsalicylic acid, indomethacin, tolazoline) do not block maturation; prostaglandins E2, D2, F2 alpha, I2, and thromboxane B2 do not induce meiosis reinitiation. On the other hand, lipoxygenase inhibitors (quercetin, butylated hydroxytoluene, and eicosatetraynoic acid) block 1-MeAde-induced maturation; although leukotrienes (A4, B4, C4, D4, E4) have no effects on oocytes, two other lipoxygenase products, 12- and 15-hydroxyeicosatetraenoic acids (and their corresponding hydroperoxy-) induce oocyte maturation (around 1 microM). The possible mode of action of the fatty acids inducing oocyte maturation is discussed.     

Glucagon and fasting do not activate peroxisomal fatty acid beta-oxidation in rat liver

In a study of the endocrine control of peroxisomes, the effects of acute glucagon treatment and fasting on hepatic peroxisomal beta-oxidation in rats have been investigated. The activity of the rate-limiting peroxisomal beta-oxidation enzyme, fatty acyl-CoA oxidase, was measured to determine whether activation of peroxisomal beta-oxidation could account for the increase in total hepatic fatty acid oxidation following acute glucagon exposure. Catalase, a peroxisomal enzyme not directly involved in beta-oxidation, was also measured as a control for total peroxisomal activity. No changes with acute glucagon treatment of intact animals were observed with either activity as measured in liver homogenates or partially purified peroxisomal fractions. These observations indicate the lack of acute control by glucagon of peroxisomal function at the level of total enzyme activity. Previous work on the effects of fasting on hepatic fatty acid beta-oxidation [H. Ishii, S. Horie, and T. Suga (1980) J. Biochem. 87, 1855-1858] suggested an enhanced role for the peroxisomal beta-oxidation pathway during starvation. It was found that the peroxisomal beta-oxidation system, as measured by fatty acyl-CoA oxidase activity, does increase with duration of fast when expressed on a per gram wet weight liver basis. However, when this activity is expressed as total liver capacity, a decline in activity with increasing duration of fast is observed. Furthermore, this decline in peroxisomal capacity parallels the decline in total liver capacity for citrate synthase, a mitochondrial matrix enzyme, and total liver protein. These data indicate that peroxisomal beta-oxidation activity is neither stimulated nor even preferentially spared from proteolysis during fasting.     

Binding of the rat liver 7-8 S dexamethasone receptor to deoxyribonucleic acid

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.     

Retinoic acid: an endogenous compound of human blood. Unequivocal demonstration of endogenous retinoic acid in normal physiological conditions.

The occurrence of retinoic acid (vitamin A acid) as a normal constituent of the vitamin A reserve from the body is demonstrated. Improved methodologies based on selected peak monitoring (spm) and high-performance liquid chromatography (hplc) are used for the detection of retinoic acid in EDTA-plasma. After extensive cleanup by double-phase extraction and chromatography on Sephadex LH-20, retinoic acid is determined as its methyl derivative by spm. A different approach using double-phase extraction combined with a preextraction step and hplc is used to confirm the findings of the spm experiments. Both techniques proved the presence of retinoic acid in human plasma at a concentration of 1 to 3 ng/ml.     

Human liver acid phosphatases: Cysteine residues of the low-molecular-weight enzyme

The stoichiometry and the reactivity of the sulfhydryl groups of a human liver acid phosphatase have been studied. The smallest (M_r = 14,400) of the three molecular-weight forms of acid phosphatase from human liver, recently purified and characterized in our laboratory, was treated with various sulfhydryl group-specific reagents: p-hydroxymercuribenzoate, p-hydroxymercuriphenylsulfonate, fluorescein mercuriacetate, methyl methanethiosulfonate, p-nitrophenoxycarbonyl methyl disulfide, and thiosulfate. A total loss of enzymatic activity was obtained in each case. By spectrophotometric titration with 5,5′-dithiobis(2-nitrobenzoate) and p-hydroxymercuriphenylsulfonate it was shown that there are six free sulfhydryls per protein molecule, consistent with the amino acid analysis of this enzyme. The same number was deduced as a result of inactivation studies carried out with p-hydroxymercuribenzoate and p-hydroxymercuriphenylsulfonate. A total loss of activity was obtained at reagent to enzyme ratios of 6:1 in both cases. Similar results were obtained upon inactivation by p-nitrophenoxycarbonyl methyl disulfide, where the enzyme was found to possess only 10% residual activity at an inhibitor-to-enzyme ratio of 6:1. With fluorescein mercuriacetate as an inactivator, total loss of activity was found at a 2.5 times molar excess of this reagent over protein. Both the stoichiometry of inactivation and fluorescence titration experiments suggest that fluorescein mercuriacetate can function as a bifunctional sulfhydryl group reagent. The activity of a totally inactivated enzyme preparation obtained following reaction with excess of p-nitrophenoxycarbonyl methyl disulfide or with methyl methanethiolsulfonate could be almost completely restored upon treatment with dithiothreitol. These data are consistent with the interpretation that in each enzyme molecule, there are six free sulfhydryl groups of almost equal reactivity, at least one of which is essential for enzymatic activity.     

Haem-binding proteins of the rat liver cytosol

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino [³h] laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [³H] haem-labelled mitochondria, [³H] haem-labelled microsomes or with [³H] haemin.These results are discussed with particular reference to ligandin.     

Isolation of copper thionein from rat liver

The inducible Cu-binding protein from adult rat liver previously referred to as Cu-chelatin has been purified and shown to be Cu-thionein. The Cu-protein was purified to homogeneity by gel filtration and thiopropyl-Sepharose chromatography. The Cu-thionein exhibited an amino acid composition similar but not identical to that of the two forms of rat liver Cd,Zn-thionein. The polypeptide-chain molecular weight of Cu-thionein was indistinguishable from that of Cd,Zn-thionein. The identification of the Cu-protein as metallothionein was substantiated by the complete immunological cross-reactivity with antisera prepared against purified rat liver Cd,Zn-thionein. Purified Cu-thionein bound 9–11 g atoms of Cu per mole of protein in an electron paramagnetic resonance nondetectable form. The $\text{Cu}\text{Zn}$ ratio of the protein is about 100. Ion-exchange chromatography resolved the Cu-protein into three polymorphic forms which differed from the polymorphism of Cd,Zn-thionein.     

N.M.R. spectroscopy and calcium binding of sialic acids: N-glycolylneuraminic acid and periodate-oxidized N-acetylneuraminic acid

N.m.r. spectroscopy (¹H- and ¹³C-) of N-glycolylneuraminic acid, and of its interaction product with Ca²⁺ at pH 7, indicated that a 1:1 complex is formed, with a formation constant of 193 M^?1 [compared to 121 M^?1 for N-acetylneuraminic acid (1)]. From analysis of electric-field shifts, an approximate position of the Ca²⁺ ion in the complex is inferred, with the hydroxyl group of the N-glycolyl group providing the additional binding. Compound 1 was oxidized with sodium periodate, and ¹³C-n.m.r. spectroscopy was applied in an attempt to identify the aldehyde formed, and to demonstrate that the loss of the glycerol-1-yl side-chain (carbon atoms 8 and 9) decreases its Ca²⁺ ion-binding capacity.     

Regulation of the betaine content of rat liver

The dietary levels of both choline and protein are major determinants of the content of betaine in rat liver. Increased protein intake decreases hepatic betaine. Our studies indicate that an increase in the betaine-homocysteine methyltransferase reaction due to an increased availability of homocysteine is the basis for this effect of dietary protein.     

Subunit composition of rat liver glutathione S-transferases

The plasmid pGTR112 contains partial coding sequences for one of the rat liver glutathione S-transferase subunits. We have used immobilized pGTR112 DNA to select for complementary and homologous liver poly(A)-RNAs under conditions of increasing stringency for hybridization. Each fraction of selected poly(A)-RNAs was assayed by in vitro translation followed by immunoprecipitation. A total of four distinct polypeptides precipitated by antiserum against rat liver glutathione S-transferases were resolved by NaDodSO₄ polyacrylamide gel electrophoresis. They are separated into two pairs according to the sequence homology of their poly(A)-RNAs with the pGTR112 DNA. Purified rat liver glutathione S-transferases can be resolved on gradient NaDodSO₄ polyacrylamide gels into four polypeptides. There should be ten isozymes of different binary combinations from four distinct subunits for the rat liver glutathione S-transferases.     

Human liver acid phosphatases: Purification and properties of a low-molecular-weight isoenzyme

A low-molecular-weight human liver acid phosphatase was purified 2580-fold to homogenity by a procedure involving ammonium sulfate fractionation, acid treatment, and SP-Sephadex ion-exchange chromatography with ion-affinity elution. The purified enzyme contains a single polypeptide chain and has a molecular weight of 14,400 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of this enzyme (E) is reported. A pH dependence study using p-nitrophenyl phosphate as a substrate (S) revealed the effect of substrate ionization (pK_a 5.2) and the participation of a group in the ES complex having a pK_a value of 7.8. The enzyme is readily inactivated by sulfhydryl reagents such as heavy metal ions. Alkylation of the enzyme with iodoacetic acid and iodoacetamide causes complete inactivation of the enzyme and this inactivation is prevented by the presence of phosphate ion. The enzyme is also inactivated by treatment with diethyl pyrocarbonate; protection against this reagent is afforded by phosphate ion. The substrate specificity of this enzyme is unusual for an acid phosphatase. Of the many alkyl and aryl phosphomonoesters tested, the only possibly physiological substrate hydrolyzed by this enzyme was flavin mononucleotide, which exhibits a V which is 3-fold larger at pH 5.0 and 6-fold larger at pH 7.0 than that for p-nitrophenyl phosphate. However, the enzyme also catalyzes the hydrolysis of acetyl phosphate at pH 5.0 with a velocity eight times larger than that reported for an acyl phosphatase from human erythrocytes.     

Degradation of rat liver metallothioneins in vitro

The degradation of zinc and cadmium-induced hepatic metallothionineins was investigated in vitro. Both zinc-thionein and cadmium-thionein were labeled in vivo with [³⁵S]cystine. The labeled proteins were isolated and purified by gel filtration and DEAE-ion exchange chromatography. Purified zinc-[³⁵S]thionein and cadmium[³⁵S]thionein were incubated with trypsin, chymotrypsin and pronase for varying times up to 24 h. The rate of degradation of zinc-thionen was twice that of cadmium-thionein when the proteins were incubated with trypsin. Virtually no digestion occurred when the proteins were incubated with chymotrypsin, whereas the rates of degradation were about equal when they were incubated with pronase. In contrast, degradation of zinc-thionein was twice that observed with cadmium-thionein when the proteins were incubated at pH 5.0 with a purified lysomal extract. Degradation of these proteins by the lysosomal proteases was 77 and 46% within 3 h for zinc-thionein and cadmium-thionein, respectively. Thionein, the metal-free from of metallothionein, was degraded extremely rapidly by both neutral and lysosomal proteases. Chromatography of the digestion products on Sephadex G-25 demonstrated that all three forms of metallothionein were degraded to species of approximately 100–300 daltons. These data indicate that metals stabilize thionein polypeptides and suggest that the degradation of metallothionein in vivo is regulated in part by the speciec of metal bound.     

Functional characterization of single-chain factor X from rat liver

¹⁴C-Labeled single-chain factor X prepared by vitamin K-dependent carboxylation in vitro was partially purified by adsorption to BaSO₄ and chromatography on DEAE-Sephacel. Known activators of factor X were analyzed for their effect on the single-chain molecule. ¹⁴C-Labeled factor X antigens were recovered immunochemically from incubation mixtures and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation with trypsin resulted in the generation of factor Xa clotting activity, and the ¹⁴C-labeled product migrated after reduction with an apparent molecular weight of 22,500 ± 1500 (mean ± 1 SD). The light chain produced by factor Xa was similar to that produced by trypsin (M_r 24,500 ± 1500; mean ± 1 SD). Incubation of single-chain factor X with factor VII and thromboplastin, factor IXa, or the factor X activating enzyme from Russell's viper venom gave a reducible product with a light chain of higher apparent molecular weight (M_r 37,000–38,000). Incubation with factor VII and thromboplastin also resulted in the generation of factor Xa clotting activity. Incubation of single-chain factor X with platelets resulted in the binding of about 20% of the ¹⁴C. The bound ¹⁴C-labeled factor X antigen released by freezing and thawing in the presence of EDTA was reduced to give a ¹⁴C-labeled polypeptide with M_r 31,000. Walker 256 tumor cells bound about 30% of the ¹⁴C. The bound material, after reduction, gave a ¹⁴C-labeled polypeptide with M_r 23,000.     

Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.