Differential effects of the diacylglycerol kinase inhibitor R59022 on thrombin versus collagen-induced human platelet secretion

R59022 is an inhibitor of the enzyme 1,2-diacylglycerol (DAG) kinase, which, by inhibiting the conversion of DAG to phosphatidic acid, causes an increase in endogenous DAG levels and the activity of the DAG-dependent enzyme protein kinase C. This property of the drug was utilized in the present study to assess the role of DAG, i.e., its relative importance as a potentiatory versus inhibitory mediator, in agonist-induced platelet activation. The phosphorylation of the 40-47-kDa protein by protein kinase C was monitored as an indicator of endogenous DAG levels and correlated with other agonist-induced platelet responses such as platelet aggregation, 5-hydroxytryptamine (5HT) secretion and arachidonate release, the agonists used being those that induce DAG formation, e.g., thrombin and collagen. Pretreatment of platelets with R59022 before agonist addition resulted in the potentiation of 5HT secretion as well as 45 kDa protein phosphorylation induced by thrombin and the DAG analogue, 1,2-dioctanoylglycerol (DiC8). However, collagen-induced 5HT secretion was significantly inhibited (70%) in the presence of R59022, which also had strong inhibitory effects on aggregation induced by collagen, as well as by thrombin and DiC8. The inhibition of collagen-induced secretion by R59022 was in contrast to the potentiatory effects of DiC8 on the same, suggesting that even although DAG acts as a potentiatory signal in this system, the inhibitory effects of R59022 on collagen-induced aggregation can mask any effects of endogenous DAG. This inhibitory effect of R59022 on agonist-induced platelet aggregation makes it unsuitable as a tool in studying the role of DAG in platelet activation induced by agonists such as collagen as well as the 'weak' agonists (ADP, adrenaline and platelet-activating factor), where aggregation mediates other responses such as arachidonate release and secretion. Furthermore, potentiatory effects of R59022 on 5HT secretion induced by phorbol 12-myristate 13-acetate and ionomycin, which are effects unlikely to be related to inhibition of DAG kinase was observed, and these effects further underline the non-specificity in the actions of R59022 and its limitations as a tool in studying platelet stimulus-response coupling.     

The diacylglycerol kinase inhibitor, R59022, potentiates cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The putative inhibitor of diacylglycerol kinase activity, 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)-ethyl-7-meth yl-5H- thiazolo[3,2-a]pyrimidin-5-one (R59022), markedly potentiated cholecystokinin-C-terminal-octapeptide(CCK-8-)stimulated enzyme secretion from isolated rabbit pancreatic acini. Maximal potentiation occurred when acini were stimulated in the presence of 5-10 microM R59022. Potentiation depended both on the concentration of R59022 and CCK-8. No potentiation was observed when acini were half-maximally stimulated, whereas the secretory response to maximal and supramaximal concentrations of secretagogue was increased by 50-60%. R59022 alone had no effect on basal enzyme secretion and the drug did not potentiate the secretory response to the Ca2+ ionophore A23187 or to the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Moreover, no increase in basal secretion was observed when acini were incubated in the presence of both R59022 and forskolin. These observations strongly suggest that receptor-mediated activation of the inositol phospholipid pathway is required for R59022-induced potentiation. R59022 inhibited the CCK-8-stimulated incorporation of 32Pi into phosphatidic acid dose dependently, without affecting the CCK-8-stimulated hydrolysis of 32P-labelled phosphatidylinositol 4,5-bisphosphate. This is consistent with an inhibitory effect of R59022 on acinar cell diacylglycerol kinase activity. The potentiating effect of R59022 was mimicked by 12-O-tetradecanoylphorbol 13-acetate added simultaneously with CCK-8. Therefore, it is concluded that in the presence of 5-10 microM R59022 the receptor-mediated increase in acinar cell diacylglycerol content is enhanced leading to enhanced activation of protein kinase C and to potentiation of the secretory response. The fact that the secretory response to maximal and supramaximal concentrations of CCK-8 is potentiated by R59022 suggests that at these concentrations of secretagogue the diacylglycerol/protein kinase C branch of the signal-transduction route is rate-limiting.     

A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets.

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.     

Partial purification and properties of diacylglycerol kinase from rat liver cytosol

Diacylglycerol:ATP kinase(EC 2.3.1.-) was highly purified (more than 2000-fold) from rat liver cytosol. The specific activity of the obtained enzyme was about 1.5 μmol phosphatidate formed/mg of protein/min. The purification procedures included ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and finally affinity chromatography on ATP-agarose. The activities of diacylglycerol:GTP kinase and monoacylglycerol:ATP kinase were copurified throughout the procedures, forming a single peak together with diacylglycerol: ATP kinase. Furthermore, these kinase activities showed a single peak when the highly purified enzyme was analyzed by a sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. The three kinase activities are, therefore, most likely catalyzed by a single enzyme. The kinase showed an apparent molecular weight of 121,000 on gel filtration and sedimented at 5.1 S in a sucrose gradient centrifugation. The apparent K_m values were 170 μm for ATP, 540 μm for GTP, and 3.0 μm for diacylglycerol. A number of nucleoside triphosphates and diphosphates competitively inhibited the kinase, in particular the activity utilizing GTP. Among the nucleotides tested, ADP was the most potent inhibitor (the apparent K_i:50 μm for diacylglycerol:ATP kinase and 42 μm for diacylglycerol:GTP kinase). The kinase required Mg²⁺ and deoxycholate for its activity, and the optimal pH was 8.0–8.5. No dependence on added phospholipids was observed.     

The diacylglycerol kinase inhibitor R59022 potentiates superoxide production but not secretion induced by fMet-Leu-Phe: effects of leupeptin and the protein kinase C inhibitor H-7

The addition of low concentrations of the chemotactic factor fMet-Leu-Phe to rabbit neutrophils in the absence of cytochalasin B produces very little superoxide. This level of superoxide can be greatly increased in neutrophils pretreated for 30 min with 10 microM of the diacyl-glycerol kinase inhibitor R59022. This potentiation occurs also in the presence of cytochalasin B. In addition, while the small level of superoxide generated by fMet-Leu-Phe is not inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7), the increase by R59022 is completely abolished by this compound. In addition, this increase can be potentiated further by leupeptin. Unlike superoxide generation, the release of lysozyme or N-acetyl-beta-glucosaminidase produced by fMet-Leu-Phe is not stimulated by R59022. The results presented here suggest that stimulation of the oxidative burst requires the generation and the maintenance of a sufficient amount of diacylglycerol and/or the rearrangement of the cytoskeleton such as the inhibition of actin polymerization. Furthermore, the membrane-associated form of protein kinase C is the one responsible for the activation of the oxidative burst. The relationship between protein kinase C activation and the stimulated oxidative burst and the physiological role of chemotactic factors in the functions of the neutrophils are discussed.     

The diacylglycerol kinase inhibitor R 59022 potentiates bombesin stimulation of protein kinase C activity and DNA synthesis in Swiss 3T3 cells

Addition of the inhibitor of diacylglycerol kinase R 59022 to quiescent Swiss 3T3 cells potentiated protein kinase C activation by the neuropeptide bombesin, a potent mitogen for these cells. This was detected as a marked shift in the dose-response relationship for bombesin-stimulated phosphorylation of a Mr 80,000 protein, which is a major, specific substrate of protein kinase C. R 59022 also promoted the inhibition of [125I]epidermal growth factor binding induced by bombesin, an effect mediated by protein kinase C. A salient feature of our results is that R 59022, at concentrations that enhanced the activation of protein kinase C in intact cells (4-6 microM), potentiated long-term mitogenesis elicited by bombesin. Thus, R 59022 may be a valuable tool for elucidating the contribution of the protein kinase C pathway in mitogenesis.     

Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two-domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N-terminal alpha/beta domain and a C-terminal two-layer beta-sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N-terminal and C-terminal domains of YegS are however counter-rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N-terminal cleft may bind activating lipids while the C-terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer-membrane, an environment that may be required for enzyme activity.     

Gene expression, cellular localization, and enzymatic activity of diacylglycerol kinase isozymes in rat ovary and placenta

Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGK, -, -, and - was detected in the ovary and placenta. Intense expression signals for DGK and - were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGK were seen more intensely in granulosa cells. In the placenta, signals for DGK and - were observed in the junctional zone, whereas those for DGK were detected in the labyrinthine zone. At higher magnification, the signals for DGK were mainly discerned in giant cytotrophoblasts, and those for DGK were found in small cytotrophoblasts of the junctional zone. DGK signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGK signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.This work was supported by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, the Uehara Memorial Foundation, the ONO Medical Research Foundation, the Ciba-Geigy Foundation (Japan) for the Promotion of Science, the Kato Memorial Bioscience Foundation, and the Yamagata Health Support Foundation (to K.G.).     

Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGK as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK translocation from the cytoplasm to the plasma membrane within 2-6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.     

Partial purification of two forms of Choline kinase and separation of Choline kinase from sphingosine kinase of rat brain

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me₂Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me₂Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me₂Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.Abbreviations Etn ethanolamine - MeEtn N-monomethylethanolamine - Me₂Etn N, N-dimethylethanolamine     

Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.     

Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.     

Cyclic AMP-dependent protein kinase in mitochondria and cytosol from different-sized follicles and corpora lutea of porcine ovaries

cAMP-dependent protein kinase was examined in mitochondria and cytosol prepared from different-sized antral follicles and corpora lutea of porcine ovaries. In all ovarian tissues examined except small follicles, protein kinase-specific activity was significantly higher in mitochondria than in cytosol, with the highest to lowest activities being found in medium (4-6 mm) follicles, large (7-12 mm) follicles, corpora lutea, and small (1-3 mm) follicles, respectively. Using the photoaffinity analogue [32P]8-N3cAMP, two major cAMP binding proteins with Mr = 47,000 (the apparent regulatory subunit of protein kinase Type I) and 54,000-56,000 (Type II) were found in all ovarian preparations. Type II was predominant in the cytosol of all ovarian samples, with the cytosolic Type I to Type II ratio increasing from approximately 0.05 in small and medium follicles top approximately 0.20 in large follicles and corpora lutea. In contrast, ovarian mitochondrial preparations contained relatively more Type I than did cytosol, with the mitochondrial Type I to Type II ratio increasing from approximately 0.50 in small and medium follicles to 0.88 in large follicles and 2.96 in corpora lutea. Also, mitochondrial [4-14C]cholesterol conversion and 3 beta-hydroxysteroid dehydrogenase/isomerase activities increased with follicle size and luteinization. These results suggest that Type I may play a role in the regulation of ovarian mitochondrial steroidogenesis.     

Purification and characterization of two isozymes of 3-phosphoglycerate kinase from the mouse.

Two isozymes of 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), designated PGK-A and PGK-B, were purified from separate extracts of muscle and testicular tissue of DBA/2J mice, respectively. A similar procedure was used to purify the corresponding isozymes from C57BL/6J mice in order to make inter-strain comparisons. The purification involved the use of affinity chromatography with an 8-(6-aminohexyl)amino-ATP-Sepharose column and DEAE-Sephadex chromatography. Lactate dehydrogenase isozyme LDH-X was also co-purified from extract of mouse testes by this two-step procedure. The same isozyme isolated from either mouse strain was found to be identical in physical and biochemical properties. Both isozymes are monomeric as determined by gel filtration chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Furthermore, the isozymes have similar molecular weights, of 47 000 +/- 2000 and exhibit similar Km values for both coenzymes and substrate, as well as temperature dependence of enzyme activity. However, it was observed that the B isozyme is more labile than the A isozyme by denaturation at high temperature, urea and acidic pH.     

Comparative studies of two cathepsin B isozymes from porcine spleen. Isolation, polypeptide chain arrangements, and enzyme specificity

Our previous studies on carbohydrate structures of purified porcine spleen cathepsin B indicated that there are two cathepsin B isozymes, each containing a different carbohydrate (Takahashi, T., Schmidt, P.G., and Tang, J. (1984) J. Biol. Chem. 259, 6059-6062). We have now isolated these two enzymes and carried out a comparative study on their structures and enzymic properties. The major isozyme (CB-I) is a two-chain enzyme (Mr = 28,000) with a light chain (Mr = 5,000) and a heavy chain (Mr = 23,000), whereas the minor enzyme (CB-II) is a single chain enzyme (Mr = 27,000). The NH2-terminal amino acid residues of CB-I were leucine and valine for the light and heavy chain, respectively. However, the NH2-terminal residue of CB-II was not available for automated Edman degradation. In addition, peptide mapping experiments indicated a difference in the primary structure of these two proteins. Despite such structural differences, they are similar in many enzymic properties. CB-I was more catalytically efficient than CB-II toward synthetic substrates, except for the substrate benzoyl-L-arginine beta-naphthylamide for which the relative catalytic efficiency is reversed. Both isozymes degraded glucagon by a dipeptidyl carboxypeptidase activity. Under the same conditions, CB-I was 4-5 times more efficient than CB-II. The results indicate that the cathepsin B isozymes are two separate gene products, but they are similar in enzymic properties.     

Arabidopsis sphingosine kinase and the effects of phytosphingosine-1-phosphate on stomatal aperture

Sphingolipids are a major component of membrane lipids and their metabolite sphingosine-1-phosphate (S1P) is a potent lipid mediator in animal cells. Recently, we have shown that the enzyme responsible for S1P production, sphingosine kinase (SphK), is stimulated by the phytohormone abscisic acid in guard cells of Arabidopsis (Arabidopsis thaliana) and that S1P is effective in regulating guard cell turgor. We have now characterized SphK from Arabidopsis leaves. SphK activity was mainly associated with the membrane fraction and phosphorylated predominantly the Delta4-unsaturated long-chain sphingoid bases sphingosine (Sph) and 4,8-sphingadienine, and to a lesser extent, the saturated long-chain sphingoid bases dihydrosphingosine and phytosphingosine (Phyto-Sph). 4-Hydroxy-8-sphingenine, which is a major sphingoid base in complex glycosphingolipids from Arabidopsis leaves, was a relatively poor substrate compared with the corresponding saturated Phyto-Sph. In contrast, mammalian SphK1 efficiently phosphorylated Sph, dihydrosphingosine, and 4,8-sphingadienine, but not the 4-hydroxylated long-chain bases Phyto-Sph and 4-hydroxy-8-sphingenine. Surface dilution kinetic analysis of Arabidopsis SphK with Sph presented in mixed Triton X-100 micelles indicated that SphK associates with the micellar surface and then with the substrate presented on the surface. In addition, measurements of SphK activity under different assay conditions combined with phylogenetic analysis suggest that multiple isoforms of SphK may be expressed in Arabidopsis. Importantly, we found that phytosphingosine-1-phosphate, similar to S1P, regulates stomatal apertures and that its action is impaired in guard cells of Arabidopsis plants harboring T-DNA null mutations in the sole prototypical G-protein alpha-subunit gene, GPA1.     

Phospholipase A2 activity on mixed lipid monolayers: Inhibition and activation of phospholipases A2 from porcine pancreas and Crotalus adamanteus by anionic and neutral amphiphiles

The effects of anionic and neutral amphiphiles on porcine pancreatic and Crotalus adamanteus phospholipases A2 were studied in a monolayer system as a function of surface pressure. The insoluble amphiphile, dicetyl phosphate (DCP), inhibited the hydrolysis of didecanoylphosphatidylcholine (DDPC) by both enzymes below their normal cutoff pressures with pure DDPC. DCP, however, enhanced enzyme penetration and thus activated the pancreatic enzyme above its normal cutoff pressure. The soluble surfactants, 3,5-dibromo- and 3,5-diiodosalicyclate, acetyl salicylate, and salicylic acid, had similar effects. 1,2-Didecanoin inhibited the hydrolysis of DDPC below the normal cutoff pressures and increased the cutoff pressures for both enzymes. Zwitterionic detergents, N-dodecyl- and N-tetradecyl-N,N-dimethyl-3-aminopropanesulfonate, were found to be potent inhibitors of the pancreatic enzyme on DDPC monolayers. Relative substrate specificities for both enzymes were determined as a function of surface pressure with phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid. Pancreatic phospholipase A2 was more active and penetrated to higher pressures with the anionic phospholipids, while the venom enzyme was more active with phosphatidylcholine.     

Characterization of two multispecific aspartate aminotransferase isozymes purified from bushbean shoots capable of transaminating aromatic amino acids and severalDL-chloro-phenylalanines

Two electrophoretically distinct isozymes ofL-phenylalanine aminotransferase (Enz I, Enz II) purified from a total soluble shoot extract of bushbean have been characterized. The M_rs of Enz I and Enz II were 100 000 and 110 000, respectively. Both isozymes showed pH optima of 8.5. Enz I was able to use either 2-oxoglutarate (2-OG) or oxaloacetate (OAA) equally as a keto acid substrate whenL-phenylalanine was the amino donor, while Enz II preferred 2-OG. Neither isozyme was able to use glyoxylate or pyruvate in the presence ofL-phenylalanine. When tested with a range of protein amino acids, both Enz I and Enz II showed the highest rate of transamination withL-aspartate, indicating that both isozymes wereL-aspartate aminotransferases capable of also showingL-aromatic aminotransferase activity.L-Phenylalanine aminotransferase activity relative toL-aspartate aminotransferase activity was found to be 0.6 % for Enz I and 3.3% for Enz II. Lineweaver-Burk plots of kinetic data gave apparent K_m values (mM) for Enz I of 2.3 (L-Asp), 55.0 (L-Phe) and 9.0 (2-OG) and for Enz II, 2.8 (L-Asp), 320.0 (L-Phe) and 8.2 (2-OG). The values were confirmed by treatment of the data by Hill plots. When tested with a series of 12 ring-substitutedDL-chlorophenylalanines, Enz I was active only with the 3-chloro- and 4-chloro-compounds, while Enz II was active with all three monochloro-compounds as well as with the 2,4-, 2,6- and 3,4-dichlorophenylalanines. The activity of Enz II with 4-chlorophenylalanine was very high, 222 % higher than that observed withDL-phenylalanine. Enz I was completely inhibited by 1.0 mM Ca²⁺ while Enz II was unaffected by this cation, which suggested different subcellular locations for each isozyme. Cell fractionation studies indicated, however, that both Enz I and Enz II were cytoplasmic. Different isozymes of this multispecific aspartate—aromatic aminotransferase were found in the chloroplasts and mitochondria of bushbean shoots.