Purification and characterization of palmitoyl-CoA ligase from rat brain microsomes

We have previously shown the existence of two separate enzymes for the synthesis of palmitoyl-CoA and lignoceroyl-CoA in rat brain microsomal membranes (1). Palmitoyl-CoA ligase activity was solubilized from brain microsomal membranes with 0.3% Triton X-100 and purified 93-fold by a combination of protein purification techniques. The Km values for the substrates palmitic acid, CoASH and ATP were 11.7 microM, 5.88 microM and 3.77 mM respectively. During activation of palmitic acid ATP is hydrolyzed to AMP and pyrophosphate, as evidenced by the inhibition of this activation by 5 mM concentrations of AMP, pyrophosphate or AMP and pyrophosphate to 70%, 60% and 85% respectively. The divalent metal ion Mg2+ was required for activity; its replacement with Mn2+ resulted in a 35% decrease in activity. Palmitoyl-CoA ligase activity was inhibited by the addition of oleic or stearic acids whereas addition of lignoceric acid or behenic acid had no effect. This supports our previous observation that palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes in rat brain microsomal membranes.     

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.     

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.     

Regulation of protein kinase C activity by cooperative interaction of Zn2+ and Ca2+

cis-Fatty acids such as oleic acid or linoleic acid have been previously shown to induce full activation of protein kinase C in the absence of Ca2+ and phospholipids (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193; Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429). In this study, we have investigated the effects of various metal ions on protein kinase C activity without the interference of Ca2+ since cis-fatty acid requires no Ca2+ for protein kinase C activation. Here we report a specific interaction of Zn2+ with protein kinase C in either a positive or negative cooperative fashion in concert with Ca2+. At low concentrations (approximately 5 microM) of Ca2+, Zn2+ enhances protein kinase C activity induced by both oleic acid and phosphatidylserine/diolein. In contrast, Zn2+ inhibits the activity at higher concentrations (over 50 microM) of Ca2+. In the absence of Ca2+, Zn2+ shows no effect on protein kinase C activity. Our results suggest that Zn2+ does not recognize or interact with protein kinase C in the absence of Ca2+, that protein kinase C possesses high and low affinity Ca2+-binding sites, and that at least one Zn2+-binding site exists which is distinct from Ca2+-binding sites.     

The effects of bovine serum albumin and oleic acid on rat pancreatic lipase and bovine milk lipoprotein lipase

The effects of bovine serum albumin on rat pancreatic lipase and bovine milk lipoprotein lipase were studied in a system of triacylglycerol emulsions stabilized by 1 1 mg/ml albumin. At concentrations greater than 1 mg/ml, albumin inhibited the activity of pancreatic lipase and interfered with enzyme binding to emulsified triacylglycerol particles. These effects could be countered by occupying five fatty acid binding sites on albumin with oleic acid. Following an initial lag period which increased with albumin concentrations, enzyme activity escaped from inhibition presumably due to saturation of fatty acid sites on albumin with oleic acid. Pancreatic lipase was active at 1 mg/ml albumin and 1 mM emulsion-bound oleic acid in the system. The effects of albumin on lipoprotein lipase were diametrically opposed to the above; enzyme activity was completely inhibited by 0.1 mM oleic acid, it increased with increasing fatty acid-free albumin concentrations and decreased as the fatty acid sites on albumin were filled. At 1 mM oleic acid and no added albumin the enzyme failed to bind at the oil water interface, whereas fatty acid-free or saturated albumin had no effect on binding. It is concluded that if the inhibition of pancreatic lipase by albumin is due to the inaccessibility of the enzyme to an oil-water interface blocked by denatured albumin, then albumin saturated with oleic acid would seem to be protected from unfolding at the interface and more readily displaced by the lipase. Pancreatic lipase and lipoprotein lipase, although sharing a number of common features, are distinct enzymes both functionally and mechanistically.     

Microsomal glycerolphosphate acyltransferase inactivation by fatty acids

Glycerolphosphate acyltransferase activity in microsomes from rat adipose tissue is shown to decrease with time upon incubation with adipose tissue cytosolic fraction. The inactivation can be prevented with serum albumin and seems to be caused by an increase in endogenous free fatty acid as a consequence of the action of cytosolic lipase(s) on the membrane lipids. Similar inactivation can be observed after short incubation of microsomes with oleic acid at micromolar concentrations. Diacylglycerol acyltransferase is also inhibited by oleic acid, although to a lesser degree. In contrast, glucose-6-phosphatase and NADPH-cytochrome reductase activities are not changed. The oleic acid effect appears to occur upon binding to the microsomal membranes and can be prevented by bovine serum albumin at protein/fatty acid molar ratios above one. These results suggest that free fatty acids may be involved in the modulation of triacylglycerol synthetic enzymes.     

Characterization of calcium-activated, phospholipid-dependent protein kinase from rat serosal mast cells and RBL-1 cells

Evidence is presented that rat serosal mast cells and cells of the rat basophilic leukemia line, RBL-1, each contain a calcium-activated, phospholipid-dependent protein kinase. The enzymes are very similar in their activation requirements to the calcium-dependent enzymes termed protein kinase Cs in brain. The enzyme is selectively stimulated by diolein and phosphatidylserine and is inhibited by several local anesthetics. The Ka for Ca2+ is 1.0 X 10(-3) M and 1.5 X 10(-4) M in mast cells and RBL-1 cells, respectively. The enzyme in mast cells is rapidly activated and apparently changed in its intracellular distribution when intact mast cells are stimulated with 48/80, A-23187, and anti-IgE and 12-O-tetradecanoyl-13-acetate in combination.     

Intestinal absorption of free oleic acid in the unanesthetized rat: evidence for a saturable component?

The intestinal absorption of free oleic acid at low intraluminal concentrations and the influence of luminal factors on its absorption were studied in the unanesthetized rat. The relationship between oleic acid concentration (30-2500 microM) and its rate of absorptions fitted best to a rectangular hyperbola (y = x/(2.19 + 0.0015x), r = 0.94). Oleic acid's rate of absorption increased as the hydrogen ion and sodium taurocholate concentrations were increased or as the thickness and resistance of the unstirred water layer were diminished or following the addition of lysolecithin. The additions of the artificial detergent Tween-80, or lecithin and linoleic, linolenic and arachidonic acids to the perfusate decreased oleic acid's rate of absorption. It was concluded that oleic acid absorption in this range of concentrations displays apparent saturation kinetics which are due to unstirred layer effects, limited aqueous solubility of oleic acid and possible saturation of cytosol fatty acid binding proteins. Factors which increase oleic acid's protonated concentration or diminish the unstirred layer resistance, enhance its absorption rate, while factors which enhance its micellar solubility or interfere with its transfer out of the cell membrane decrease its overall rate of absorption.     

Response of three enzymes to oleic acid, trypsin, and calmodulin chemically modified with a reactive phenothiazine

Calmodulin was covalently modified with 10-(1-propionyloxysuccinimide)-2-trifluoromethylphenothiazine++ + to stoichiometries between 0 and 2 mol/mol in the presence of Ca2+. The modified calmodulins, oleic acid, and trypsin were assayed for their ability to activate pea plant NAD kinase, bovine brain 3',5'-cAMP phosphodiesterase, and human erythrocyte Ca2+-ATPase. All modified calmodulins activated both phosphodiesterase and Ca2+-ATPase; at the highest concentration assayed, calmodulin modified with 2 mol of reagent/mol activated phosphodiesterase and Ca2+-ATPase to 53% and 100%, respectively, of the activation obtained with unmodified calmodulin. However, higher concentrations of the modified calmodulins were required to observe the same activation; at least 900-fold and 100-fold higher concentrations were required for the two enzymes, respectively. NAD kinase was not activated by any calmodulin labeled to a stoichiometry greater than 1 mol/mol even when a concentration equal to 17,000 times the apparent dissociation constant of calmodulin for NAD kinase was assayed. Therefore, the modified protein (and not some fraction resistant to labeling) is active toward the mammalian enzymes but inactive toward plant NAD kinase. The different response of the three enzymes to the chemical modification suggests that the enzymes may utilize different binding domains on calmodulin. NAD kinase also was not activated by other known activators of the two mammalian enzymes, namely lipids and limited proteolysis. In parallel experiments using the same agents on each enzyme, NAD kinase was the only enzyme of the three that was not activated by oleic acid and several other lipids or by limited trypsin digestion. These results show that NAD kinase possesses several attributes which would not be predicted by current models of the mechanism of activation of enzymes by calmodulin.     

Unesterified long-chain fatty acids inhibit thyroid hormone binding to the nuclear receptor. Solubilized receptor and the receptor in cultured cells

Unesterified long-chain fatty acids strongly inhibited thyroid hormone (T3) binding to nuclear receptors extracted from rat liver, kidney, spleen, brain, testis and heart. Oleic acid was the most potent inhibitor, attaining 50% inhibition at 2.8 microM. Oleic acid similarly inhibited the partially purified receptor and enhanced dissociation of the preformed T3-receptor complex. The fatty acid acted in a soluble form and in a competitive manner for the T3-binding sites, thereby reducing the affinity of the receptor for T3. The affinity of the receptor for oleic acid (Ki) was 1.0 microM. In HTC rat hepatoma cells in culture, fatty acids added to the medium reached the nucleus and inhibited nuclear T3 binding; oleic acid being the most potent. T3 binding of the cells was reversibly restored in fresh medium free of added fatty acids. Oleic acid did not affect all the T3-binding sites in the HTC cells: one form (80%) was inhibited and the other was not and these two forms were commonly present in all rat tissues examined. Thus, fatty acids inhibited the solubilized nuclear receptor as well as a class of nuclear T3-binding sites in cells in culture.     

Mode of activation of bovine brain inositol 1,4,5-trisphosphate 3-kinase by calmodulin and calcium.

The effect of Ca2+ and calmodulin (CaM) on the activation of purified bovine brain Ins(1,4,5)P3 kinase was quantified and interpreted according to the model of sequential equilibria generally used for other calmodulin-stimulated systems. Two main conclusions can be drawn. (i) CaM.Ca3 and CaM.Ca4 together are the biologically active species in vitro, as is the case for the great majority of other calmodulin targets. (ii) These species bind in a non-co-operative way to the enzyme with an affinity constant of 8.23 x 10(9) M-1, i.e. approx 10-fold higher than for most calmodulin-activated target enzymes. The dose-response curve of the activation of Ins(1,4,5)P3 kinase by calmodulin is not significantly impaired by melittin and trifluoperazine, whereas under very similar assay conditions the half-maximal activation of bovine brain cyclic AMP phosphodiesterase requires over 30-50-fold higher concentrations of CaM when 1 microM melittin or 20 microM-trifluoperazine is present in the assay medium. Similarly, 1 microM of the anti-calmodulin peptides seminalplasmin and gramicidin S, as well as 20 microM of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W7), do not inhibit the activation process. These data suggest that binding and activation of Ins(1,4,5)P3 kinase require surface sites of calmodulin which are different from those involved in the binding of most other target enzymes or of model peptides.     

Calcium-activated phospholipid-dependent protein kinases from rat liver: characterization of purified isoenzymic forms

1. This report describes characteristics of the three isoenzymic forms of protein kinase C purified in our laboratory from rat liver. 2. All three C-kinases phosphorylated several histone preparations, and to a much lesser degree, other protein substrates and had similar Km values for ATP and histones. 3. Each isoenzyme demonstrated an absolute requirement for Ca2+ and negatively charged phospholipids. 4. Among various phospholipids tested, phosphatidylserine from bovine brain was most effective with approximately 220 fold stimulation over basal activity. 5. Both diolein and 12-O-tetradecanoylphorbol-13-acetate decreased the Ca2+ requirement of the isoenzymes and also directly stimulated C-II and C-III enzymes in the presence of suboptimal concentrations of Ca2+ and phosphatidylserine.     

Effect of oleic acid on mitochondrial oxidative phosphorylation in rat brain slices

We tested the effect of oleic acid on oxidative phosphorylation and free fatty acid composition in rat brain slices simultaneously to investigate the relationship between the change in respiratory control ratio and the uptake of oleic acid in the brain mitochondria. The uncoupling of mitochondria was observed when the ratio of oleic acid to stearic acid in the free fatty acid fraction was nearly doubled, but was not recovered even by the addition of fatty acid-free bovine serum albumin. The data suggest that the intactness of oxidative phosphorylation of brain mitochondria is maintained by the precise control of the free fatty acid composition in the mitochondrial membranes.     

Biosynthesis of platelet-activating factor in glandular gastric mucosa. Evidence for the involvement of the ''de novo'' pathway and modulation by fatty acids.

The biosynthesis of platelet-activating factor (PAF), a phospholipid autocoid with potent ulcerogenic properties that is produced in secretory exocrine glands by physiological secretagogues, was assessed in microsomal preparations of glandular gastric mucosa. For this purpose, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67); the enzymes of the 'de novo' pathway: 1-O-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP):acetyl-CoA acetyltransferase and 1-O-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-choline cholinephosphotransferase (EC 2.7.8.16); and some enzymes involved in the catabolism of PAF and lyso-PAF were assayed. Only the enzymes of the 'de novo' pathway and small amounts of PAF acetylhydrolase, phospholipase A2 and a lysophospholipase D acting on either lipids could be detected in the gastric preparations, whereas lyso-PAF:acetyl-CoA acetyltransferase activity was undetectable. The specific activity of alkyl-lyso-GP:acetyl-CoA acetyltransferase in the gastric mucosa was about one-tenth of that found in spleen microsomes and its apparent Km for acetyl-CoA was 454 microM compared with 277 microM in spleen microsomes. Glandular mucosa homogenates contained preformed PAF at a concentration of 2.7 +/- 0.7 ng equivalents of PAF (hexadecyl)/mg of protein. When gastric microsomes were incubated with micromolar concentrations of fatty acids (arachidonic, palmitic and oleic) prior to the assay of dithiothreitol (DTT)-insensitive cholinephosphotransferase, a dose-dependent reduction in the formation of PAF was observed, arachidonic acid being the most potent inhibitor, followed by linoleic acid (only tested on spleen microsomes) and oleic acid. By contrast, 1,2-diolein and phosphatidylcholine (dipalmitoyl) showed no or little effect. These results indicate that glandular gastric mucosa can produce PAF through the 'de novo' pathway, and that fatty acids, especially unsaturated, can reduce that synthesis by modulating the expression of DTT-insensitive cholinephosphotransferase.     

Interaction of tyrosine hydroxylase with ribonucleic acid and purification with DNA-cellulose or poly(A)-sepharose affinity chromatography

Tyrosine hydroxylase in bovine adrenal medulla was activated up to fourfold by incubation with low concentrations (15 micrograms/ml) of ribonucleic acids. At higher RNA concentrations, enzyme activity was inhibited. This interaction with RNA was exploited with the use of poly(A)-Sepharose and DNA-cellulose to effect a rapid purification of stable tyrosine hydroxylase from rat brain and bovine adrenal medulla in high yield (up to 58%). With the purified rat brain enzyme, RNA acted as an uncompetitive inhibitor, a concentration of 15 micrograms/ml lowering the Vmax of tyrosine hydroxylase from 1050 to 569 nmol min-1 mg-1 and lowering the Km for tyrosine from 6.1 to 3.6 microM. With the natural cofactor, tetrahydrobiopterin (BH4), two Km values were obtained, indicating the presence of two forms of the enzyme. Both Km values were decreased only slightly by RNA. The purified brain and adrenal enzymes both contained about 0.07 mol of phosphate/63,000-Da subunit; in both cases, cyclic AMP-dependent protein kinase catalyzed the incorporation of an additional 0.8 mol of phosphate/subunit. The purified enzyme also contains ribonucleic acid, which comprises about 10% of the total mass and appears to be important for full activity.     

Structural studies on bovine spleen heme oxygenase. Immunological and structural diversity among mammalian heme oxygenase enzymes.

Heme oxygenase is an Mr 32,000 microsomal enzyme which catalyzes the rate-limiting step in the oxidative catabolism of heme to yield equimolar quantities of biliverdin IX alpha, carbon monoxide, and iron. In the present investigation, evidence is presented suggesting that immunochemical and structural differences exist between bovine spleen heme oxygenase and heme oxygenase enzymes from other mammalian species. Using an antibody directed against bovine spleen heme oxygenase, enzyme-linked immunosorbent assays, Western blotting experiments, and cell-free translation immunoprecipitation studies showed that bovine spleen heme oxygenase is only weakly immunochemically related to heme oxygenase from rat spleen. This observation was supported by the fact that a rat spleen heme oxygenase cDNA probe did not hybridize significantly to bovine spleen heme oxygenase mRNA in Northern analyses nor to restriction fragments containing the bovine heme oxygenase gene in Southern analyses. Tryptic peptides were prepared from bovine spleen heme oxygenase and the amino acid sequences of nine peptides comprising 94 amino acid residues were determined, providing the first information on the primary structure of bovine spleen heme oxygenase. Comparison of the sequences of these tryptic peptides with regions of the deduced amino acid sequences of rat spleen and human macrophage heme oxygenase revealed sequence similarities ranging from 55 to 100%. Several peptides displaying the highest degree of sequence similarity were found to occur in regions of the heme oxygenase molecule postulated to contain the heme binding site, indicating that despite the immunochemical and apparent structural differences between bovine spleen heme oxygenase and the rat and human enzymes, functionally important amino acid residues have been conserved in the evolution of mammalian heme oxygenase genes.     

Phospholipid and calmodulin activation of solubilized calcium-transport ATPase from human erythrocytes: regulation by magnesium

The effect of phospholipids on Triton X-100 solubilized (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes has been examined. The enzyme activity was increased by phosphatidylinositol, phosphatidylserine, and phosphatidic acid at both low (2 micrometer) and high (65 micrometer) free Ca2+ concentrations, while phosphatidylcholine had little effect and phosphatidylethanolamine and cardiolipin inhibited the (Ca2+ + Mg2+)-ATPase activity at all Ca2+ concentrations studied. The diacylglycerol, diolein, inhibited the enzyme at high, but not low, Ca2+ concentrations. Low concentrations of phospholipase A2 (1-2 international units) also activated the solubilized enzyme, at least in part by releasing free fatty acids, as the activation was mimicked by oleic acid (1-2 mumol/mg protein) and was abolished by fatty acid depleted bovine serum albumin. The combined activation by saturating levels of phosphatidylserine and calmodulin was additive at 6.5 mM MgCl2, and probably occurred at distinct sites on a regulatory component of the enzyme. The activation by both effectors was antagonized by MgCl2 at similar concentrations. Analysis of various models suggested that phosphatidylserine had two effects on (Ca2+ + Mg2+)-ATPase activity. First, a low Ca2+ affinity form of the enzyme was converted to a high Ca2+ affinity form, which was more sensitive to Ca2+ inhibition. Second, it increased the turnover of the enzyme, probably by enhancing its dephosphorylation, which was mimicked in this study by the Ca2+-dependent p-nitrophenylphosphatase partial reaction.     

Isorenin, pseudorenin, cathepsin D and renin. A comparative enzymatic study of angiotensin-forming enzymes

1. Renin was purified 30 000-fold from rat kidneys by chromatography on DEAE-cellulose and SP-Sephadex, and by affinity chromatography on pepstatinyl-Sepharose. 2. The enzymatic properties of isorenin from rat brain, pseudorenin from hog spleen, cathepsin D from bovine spleen, and renin from rat kidneys were compared: Isorenin, pseudorenin and cathepsin D generate angiotensin from tetradecapeptide renin substrate with pH optima around 4.9, renin at 6.0. With sheep angiotensinogen as substrate, isorenin, pseudorenin and cathepsin D have similar pH profiles (pH optima at 3.9 and 5.5), in contrast to renin (pH optimum at 6.8). 3. The angiotensin-formation from tetradecapeptide by isorenin, pseudorenin and cathepsin D was inhibited by albumin, alpha-and beta-globulins. These 3 enzymes have acid protease activity at pH 3.2 with hemoglobin as the substrate. Renin is not inhibited by proteins and has no acid protease activity. 4. Renin generates angiotensin I from various angiotensinogens at least 100 000 times faster than isorenin, pseudorenin or cathepsin D, and 3000 000 times faster than isorenin when compared at pH 7.2 with rat angiotensinogen as substrate. 5. The 3 'non-renin' enzymes exhibit a high sensitivity to inhibition by pepstatin (Ki less than 5.10(-10) M), in contrast to renin (Ki approximately 6-10(-7) M), at pH 5.5. 6. It is concluded from the data that isorenin from rat brain and pseudorenin from hog spleen are closely related to, or identical with cathepsin D.