Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity.

The first step in the production of eicosanoids and platelet-activating factor is the hydrolysis of arachidonic acid from membrane phospholipid by phospholipase A2. We previously purified from the macrophage cell line RAW 264.7 an intracellular phospholipase A2 that preferentially hydrolyzes sn-2-arachidonic acid. The enzyme exhibits a molecular mass of 100 kDa and an isoelectric point of 5.6. When assayed for other activities, the phospholipase A2 was found to exhibit lysophospholipase activity against palmitoyllysoglycerophosphocholine, and both activities copurified to a single band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. An antibody against the macrophage enzyme was found to quantitatively immunoprecipitate both phospholipase A2 and lysophospholipase activities from a crude cytosolic fraction. When the immunoprecipitated material was analyzed on immunoblots, a single band at 100 kDa was evident, further suggesting that a single protein possessed both enzyme activities. When assayed as a function of palmitoyllysoglycerophosphocholine concentration and plotted as a double-reciprocal plot, two different slopes were apparent, corresponding to concentrations above and below the critical micellar concentration (7 microM) of the substrate. Above the critical micellar concentration, lysophospholipase exhibited an apparent Km of 25 microM and a Vmax of 1.5 mumol/min/mg. Calcium was not required for lysophospholipase activity, in contrast to phospholipase A2 activity. The enzyme, when assayed as either a phospholipase A2 or lysophospholipase, exhibited nonlinear kinetics beyond 1-2 min despite low substrate conversion. Readdition to more substrate after the activity plateaued did not result in further enzyme activity, ruling out substrate depletion. Readdition of enzyme, however, resulted in another burst of enzyme activity. The results are not consistent with product inhibition, but suggest that the enzyme may be subject to inactivation during catalysis.     

Studies on lysophospholipases. IV. The subcellular distribution of two lysolecithin-hydrolyzing enzymes in beef liver.

1. In a previous paper (Biochim. Biophys. Acta (1974) 369, 50-63) the purification of two proteins with lysophospholipase activity (EC 3.1.1.5), provisionally denoted lysophospholipase I and lysophospholipase II, has been described. The subcellular localization of both enzymes was investigated by cell fractionation studies. 2. For each subcellular fraction the total lysophospholipase activity, after solubilization by n-butanol treatment, was separated into a lysophospholipase I and II contribution by DEAE-Sephadex ion exchange chromatography. 3. Lysophospholipase I was found to be a soluble enzyme with a bimodal distribution. Highest relative specific activities were measured in the mitochondrial and the cytoplasmic fraction. Evidence is presented indicating that this enzyme is present in the mitochondrial matrix fraction. 4. Lysophospholipase II appeared to be a membrane-bound enzyme with highest relative specific activity in the microsomal fraction.     

Equal inhibition of HIV replication by stereoisomers of phosphatidyl-azidothymidine. Lack of stereospecificity of lysosomal phospholipase A1.

Glycerol-1-P and glycerol-3-P stereoisomers of dipalmitoylphosphatidylazidothymidine were synthesized and found to have equal antiretroviral activity in HIV-infected HT4-6C cells. It was anticipated that the glycerol-1-P isomer would be less active because of slow metabolic conversion by cellular phospholipases A and C, but the antiretroviral results suggested that the human cell line (HT4-6C) may have phospholipases capable of hydrolyzing 2,3-dipalmitoyl-sn-glycerol-1-phospho-5'-azidothymidine (AZT). To evaluate this possibility, we purified lysosomal phospholipase A1, an enzyme known to play a major role in cellular phospholipid catabolism. This enzyme rapidly hydrolyzed both the sn-1 and sn-3 isomers of dipalmitoylphosphatidyl-AZT. We synthesized sn-2,3-dipalmitoyl-glycero-1-phosphocholine and found that it is also hydrolyzed readily by lysosomal phospholipase A1 although the Vmax, 59 mumol mg-1 h-1, is slightly lower than that of the sn-1,2-dipalmitoyl-glycero-3-phosphocholine, 89 mumol mg-1 h-1. In conclusion, our studies show that sn-2,3-dipalmitoyl-glycerol-1-phospho-AZT is equal in antiviral activity to sn-1,2-dipalmitoyl-glycero-3-phospho-AZT in HIV-infected HT4-6C cells. This surprising result is due in part to the lack of stereospecificity of lysosomal phospholipase A1.     

Purification and characterization of a lysophospholipase from human amnionic membranes

We have identified the presence of a lysophospholipase in human placental tissues and have purified this enzyme from the amnion. The specific activity was highest in the amnion and decreased across adjacent tissues. The purification involved the use of DEAE-Sephadex, phenyl-Sepharose, hydroxylapatite, and sulfylpropyl Sephadex chromatography. The activity of the purified enzyme toward palmitoyl lysophosphatidylcholine is 2.5 mumol min-1 mg-1 and the pH optimum is 7.0. The enzyme is not inhibited by EDTA and does not appear to have a metal ion requirement. The enzyme may be of membrane origin; the purified enzyme requires the presence of detergent during storage. The effects of substrate composition and physical state on enzymatic activity were explored. The enzyme was not active toward mono-, di-, or triglycerides, nor toward diacyl phospholipid. The enzyme was active toward myristoyl and palmitoyl lysophosphatidylcholine at concentrations where these substrates spontaneously form micelles or where Triton X-100 was used to induce co-micellization of the substrate at low concentrations with detergent. A role for this enzyme in processing the lysophospholipid product of phospholipase A action must be considered in evaluating arachidonic acid production in human fetal membranes and placental tissue, particularly during the initiation of labor.     

Inhibition of lysophospholipase by cholesterol in rabbit aorta

Lysophospholipase activity was measured in rabbit aorta using 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine as a substrate. The enzyme did not require Ca2+ for its activation and the maximal activation was attained in the presence of EGTA. Cholesterol dose-dependently inhibited the lysophospholipase activity in the soluble fraction and IC50 value was approximately 15 microM. Lineweaver-Burk plot revealed that cholesterol competitively inhibited lysophospholipase and Km values in the presence and absence of cholesterol (15.5 microM) were 12.3 and 2.8 microM, respectively. Vmax values were approximately 475 pmol/min.mg. The results suggest that cholesterol can interact with the enzyme per se, resulting in the inhibition of the lysophospholipase activity in rabbit aorta.     

Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain

The coupling of various agonist receptors to the hydrolysis of phosphoinositides has generated much interest in the nature of the phospholipase C that is activated. Here we report the purification of a bovine brain phospholipase C derived from the particulate fraction. A 1000-fold purification was achieved by a combination of heparin-Sepharose, DEAE-cellulose and gel-permeation chromatography. The purified enzyme appears to be monomeric and under denaturing conditions shows a single staining major polypeptide of molecular mass 154 kDa in SDS gels. The enzyme is specific for phosphoinositides although it shows a marked preference for the polyphosphoinositides. With phosphatidylinositol 4,5-bisphosphate as substrate the enzyme expresses a specific activity of greater than 100 mumol min-1 mg-1. The phospholipase C is activated by Ca2+ (0.1-10 microM). The behaviour of this particulate enzyme is discussed in the context of a agonist-induced phosphatidylinositol hydrolysis.     

Analysis of human synovial fluid phospholipase A2 on short chain phosphatidylcholine-mixed micelles: development of a spectrophotometric assay suitable for a microtiterplate reader.

The development of a reliable assay for human synovial fluid phospholipase A2 (HSF PLA2) is important for the kinetic characterization of the enzyme and for the identification of enzyme inhibitors. This enzyme behaves differently from other extracellular PLA2s in many standard phospholipase assays and is generally assayed using radiolabeled, autoclaved Escherichia coli as a substrate. We have now developed a nonradioactive, continuous, spectrophotometric assay for this enzyme that is adaptable for use with a microtiterplate reader and is suitable for screening enzyme inhibitors. The assay uses a thioester derivative of diheptanoyl phosphatidylcholine as a substrate, with which the enzyme displays a specific activity of about 25 mumol min-1 mg-1. The substrate concentration curve fits a Hill equation with an apparent Km of 500 microM and a Hill coefficient of two. The enzyme has a pH optimum of 7.5 in this assay and requires about 10 mM Ca2+ for maximal activity. The presence of 0.3 mM Triton X-100 was necessary to solubilize the substrate; however, higher concentrations of the detergent inhibited enzyme activity. Using this spectrophotometric assay, inhibition of HSF PLA2 by a thioether phosphonate phosphatidylethanolamine analog was observed with an IC50 of 18 microM.     

Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques

1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate containing lysophospholipase II in enzymically active form could be isolated. 3. Microsomal lysophospholipase activity was completely inhibited by [3H]diisopropylphosphofluoridate. Enzyme labelled in this way was isolated by immunoprecipitation from control and chymotrypsin-treated microsomes. Sodium dodecyl sulfate disc gel electrohporesis of the immunoprecipitates showed that chymotrypsin treatment of intact microsomes had no influence on the molecular weight of the enzyme. 4. Attempts to label the lysophospholipase II in microsomes by lactoperoxidase catalyzed iodination or by reaction with the diazonium salt of [125I]iodosulfanilic acid were negative, although both techniques labelled other microsomal proteins efficiently. 5. Antibody absorption experiments gave no indication for the presence of lysophospholipase antigenic sites on the outside surface of microsomes. 6. These experiments are interpreted to indicate that lysophospholipase II is exclusively located at the luminal side of the microsomal membrane.     

Solubilization, purification, and characterization of a membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line

The release of free arachidonic acid from membrane phospholipids is believed to be the rate-controlling step in the production of the prostaglandins, leukotrienes, and related metabolites in inflammatory cells such as the macrophage. We have previously identified several different phospholipases in the macrophage-like cell line P388D1 potentially capable of controlling arachidonic acid release. Among them, a membrane-bound, alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular interest because of the likelihood that the regulatory enzyme has these properties. This phospholipase A2 has now been solubilized from the membrane fraction with octyl glucoside and partially purified. The first two steps in this purification are butanol extractions that yield a lyophilized, stable preparation of phospholipase A2 lacking other phospholipase activities. This phospholipase A2 shows considerably more activity when assayed in the presence of glycerol, regardless of whether the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated vesicles or mixed micelles with the nonionic surfactant Triton X-100. Glycerol (70%) increases both the Vmax and the Km with both substrate forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater for vesicles than for mixed micelles. The lyophilized preparation of the enzyme is routinely purified about 60-fold and is suitable for evaluating phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps in the purification are acetonitrile extraction followed by high performance liquid chromatography on an Aquapore BU-300 column and a Superose 12 column. This yields a 2500-fold purification of the membrane-bound phospholipase A2 with a 25% recovery and a specific activity of about 800 nmol min-1 mg-1 toward 100 microM dipalmitoylphosphatidylcholine in mixed micelles. When this material was subjected to analysis on a Superose 12 sizing column, the molecular mass of the active fraction was approximately 18,000 daltons.     

Phospholipases of Leptospira. I. Presence of phospholipase A1 and lysophospholipase in Leptospira biflexa

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro. Phospholipase A₁ was identified by the formation of ³²P- and ¹⁴C-labeled lyso-derivatives from ³²P-phosphatidylcholine, ³²P-phosphatidylethanolamine, or 1-acyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphorylcholine. Phospholipase A₁ activity was independent of lipase in the microorganism since ¹⁴C-labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed. Lysophospholipase activity was also demonstrated using ³²P- and non-labeled lysophosphatidylcholine. The activity of phospholipase A₁ was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat. Phospholipase C activity, however, could not be detected because no radioactive di- and monoacylglycerol was found in the experiment with 1-acyl-2-[1-¹⁴C]-oleoyl-sn-glycero-3-phosphorylcholine as the substrate. It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A₁ and/or A₂?) and lysophospholipase to glycerophosphorylethanolamine via 2-acyl-type-lyso-derivative as one metabolic pathway of the substrate.     

A novel inositol-phospholipid-specific phospholipase C. Rapid purification and characterization

A novel bovine brain inositol-phospholipid-specific phospholipase C has been identified on the basis of chromatographic behaviour and purified to apparent homogeneity by a rapid three-step procedure. The purified enzyme has a molecular mass of 85 kDa on SDS/polyacrylamide gel electrophoresis and a specific activity of 24 mumol.min-1.mg-1. The enzyme is dependent on Ca2+ and shows a marked preference for inositol phospholipid substrates. The unique nature of this polypeptide was confirmed through partial protein sequence analysis.     

Purification of bovine brain inositol-1,4,5-trisphosphate 5-phosphatase.

In bovine brain, two soluble inositol-1,4,5-trisphosphate (InsP3) 5-phosphatases, which catalyse the dephosphorylation of InsP3 to inositol 1,4-bisphosphate, have been separated by DEAE-Sephacel. Type I, i.e. the first eluted enzyme, is the main soluble form and is reminiscent of the membrane-bound enzyme by multiple criteria. Type I was purified to apparent homogeneity by a method involving chromatography on DEAE-Sephacel, Blue-Sepharose, Sephacryl S-200, phosphocellulose, and C18 HPLC. A single protein band of 42-43 kDa was identified by SDS/PAGE, corresponding to the peak of maximal activity. InsP3 5-phosphatase was purified to apparent homogeneity to a final yield of 45-50 micrograms protein. The minimal estimate value of the Vmax for InsP3 5-phosphatase was in the range 20-35 mumol.min-1.mg protein-1.     

Isolation and properties of glycerol-3-phosphate oxidoreductase from human placenta

Glycerol-3-phosphate oxidoreductase (sn-glycerol 3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from human placenta has been purified by chromatography on 2,4,6-trinitrobenzenehexamethylenediamine-Sepharose, DEAE-Sephadex A-50 and 5'-AMP-Sepharose 4B approximately 15800-fold with an overall yield of about 19%. The final purified material displayed a specific activity of about 88 mumol NADH min-1 mg protein-1 and a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The native molecular mass, determined by Ultrogel AcA 44 filtration, was 62000 +/- 2000 whereas the subunit molecular mass, established on polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate, was 38000 +/- 500. The isoelectric point of the enzyme protein, determined by column isoelectric focusing, was found to be 5.29 +/- 0.09. The pH optimum of the placental enzyme was in the range 7.4-8.1 for dihydroxyacetone phosphate reduction and 8.7-9.2 for sn-glycerol 3-phosphate oxidation. The apparent Michaelis constants (Km) for dihydroxyacetone phosphate, NADH, sn-glycerol 3-phosphate and NAD+ were 26 microM, 5 microM, 143 microM and 36 microM respectively. The activity ratio of cytoplasmic glycerol-3-phosphate oxidoreductase to mitochondrial glycerol-3-phosphate dehydrogenase in human placental tissue was 1:2. The consumption of oxygen by human placental mitochondria incubated with the purified glycerol-3-phosphate oxidoreductase, NADH and dihydroxyacetone phosphate was similar to that observed in the presence of sn-glycerol 3-phosphate. The possible physiological role of glycerol-3-phosphate oxidoreductase in placental metabolism is discussed.     

Studies on the transverse localization of lysophospholipase in bovine liver microsomes using proteolytic enzymes.

1. Sonication of bovine liver microsomes completely solubilized the membrane-bound lysophospholipase II (EC 3.1.1.5). Co-chromatography with purified 125I-labelled lysophospholipase indicated that the enzyme was solubilized from microsomes in a lipid-free state. 2. In the presence of residual microsomal membranes, the solubilized lysophospholipase could only be partly degraded by trypsin (EC 3.4.21.4). Therefore, trypsin could not be used to study the transmembrane disposition of lysophospholipase in intact microsomes. 3. Chymotrypsin (EC 3.4.21.1) destroyed the solubilized lysophospholipase activity, even in the presence of residual microsomal membranes. 4. Lysophospholipase in intact microsomal vesicles was resistant to chymotrypsin digestion. 5. When microsomal vesicles were made leaky with lysophosphatidylcholine, chymotrypsin destroyed more than 95% of the lysophospholipase activity. 6. It is concluded from these experiments that at least the active center of lysophospholipase is located at the luminal side of the bovine liver microsomal membrane.     

Subcellular localization of the phospholipases A of rat heart: evidence for a cytosolic phospholipase A1

During myocardial ischemia increased levels of lysoglycerophospholipids have been reported which may be deleterious to myocardial function. Phospholipases are presumed to be important in the regulation of this process. To further quantify and characterize the activity of heart phospholipases, we carried out a systematic analysis of phospholipase A activity in rat heart subcellular fractions isolated by the method of Palmer et al. (J. Biol. Chem. 1972. 262: 8731-8739). Neutral phospholipase A was recovered predominately in the cytosolic (soluble) fraction which represented 46% of recovered activity, while the microsomal and subsarcolemmal mitochondrial fractions represented 15% and 12% of the total recovered activity, respectively. Cytosolic phospholipase A differed from the two principal membrane-bound phospholipases A in its pH dependence and apparent Km for substrate. The cytosolic enzyme had a Km (apparent) for dioleoylphosphatidylcholine of 0.07 mM versus 0.28-0.33 mM for the membrane-associated phospholipases A. Acid phospholipase A activity had a subcellular distribution consistent with a lysosomal localization. Lysophospholipase was found principally in the cytosolic, microsomal, and the subsarcolemmal and interfibrillar mitochondrial fractions where it represented 46, 17, 6.3, and 6.9% of the recovered activity, respectively. The positional specificity of the respective phospholipases was assessed. This analysis was complicated by the fact that in heart, lysophospholipase has an observed Vmax 3.6- to 4.5-fold greater than that of phospholipase A in the various subcellular fractions. Equations were derived to obtain corrected values for the activity of phospholipases A1 and A2. Using this method we found that the cytosolic and lysosomal fractions contained phospholipase A1, while the mitochondrial fractions contained primarily phospholipase A2. In heart microsomes, the positional specificity of phospholipase A could not be determined because lysophospholipase activity was very high and lysophosphatidylcholine did not accumulate.     

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.     

Purification and characterization of phospholipase A2 released from rat platelets

It was found that phospholipase A2 and lysophospholipase, both of which were released from thrombin-stimulated rat platelets, had high affinity to insolubilized heparin. Phospholipase A2 released from rat platelets was purified by the sequential use of column chromatography on heparin-Sepharose and TSK gel G2000SW (high-performance liquid chromatography, HPLC). The enzyme was near homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, and its Mr was estimated to be 13,500. The purified enzyme was labile and lost its activity within 1 h when incubated at 37 degrees C. Phospholipids or detergent in the solution protected the enzyme against inactivation. Phospholipase activity was inhibited by p-bromophenacylbromide, but not by diisopropylfluorophosphate or iodoacetamide. Lysophospholipase, which was also released from rat platelets, was separated from phospholipase A2 by chromatography on heparin-Sepharose.     

Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis.

Two lysophospholipases were isolated from the venom of an Australian elapid snake (subfamily Acanthophiinae), Pseudechis australis, by sequential chromatography on CM-52 cellulose, Sephadex G-75 and DE-52 cellulose columns. They were very similar to each other. One of them, lysophospholipase I, was obtained as a homodimer, the monomer of which consisted of 123 amino acid residues with seven disulphide bridges. The amino acid composition and the N-terminal amino acid sequence of the enzyme were similar to those of phospholipase A2, Ca2+ was required for its activity and the maximum activity was attained at 2 mM-CaCl2 in the presence of 1 mM-EDTA. The optimum pH was 7.5. Lysophospholipase I hydrolysed lysophosphatidylcholine more rapidly than lysophosphatidylethanolamine. It did not hydrolyse, however, phosphatidylcholine, 1-palmitoylglycerol, tripalmitoylglycerol or p-nitrophenyl acetate. Modification of the enzyme with p-bromophenacyl bromide or 2-nitrophenylsulphenyl chloride suppressed the activity. A strong direct haemolytic activity was exhibited when the lysophospholipase was present together with phospholipase A2.     

Substrate specificity of a multifunctional calmodulin-dependent protein kinase

The substrate specificity of the multifunctional calmodulin-dependent protein kinase from skeletal muscle has been studied using a series of synthetic peptide analogs. The enzyme phosphorylated a synthetic peptide corresponding to the NH2-terminal 10 residues of glycogen synthase, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH2, stoichiometrically at Ser-7, the same residue phosphorylated in the parent protein. The synthetic peptide was phosphorylated with a Vmax of 12.5 mumol X min-1 X mg-1 and an apparent Km of 7.5 microM compared to values of 1.2 mumol X min-1 X mg-1 and 3.1 microM, respectively, for glycogen synthase. Similarly, a synthetic peptide corresponding to the NH2-terminal 23 residues of smooth muscle myosin light chain was readily phosphorylated on Ser-19 with a Km of 4 microM and a Vmax of 5.4 mumol X min-1 X mg-1. The importance of the arginine 3 residues NH2-terminal to the phosphorylated serine in each of these peptides was evident from experiments in which this arginine was substituted by either leucine or alanine, as well as from experiments in which its position in the myosin light chain sequence was varied. Positioning arginine 16 at residues 14 or 17 abolished phosphorylation, while location at residue 15 not only decreased Vmax 14-fold but switched the major site of phosphorylation from Ser-19 to Thr-18. It is concluded that the sequence Arg-X-Y-Ser(Thr) represents the minimum specificity determinant for the multifunctional calmodulin-dependent protein kinases. Studies with various synthetic peptide substrates and their analogs revealed that the specificity determinants of the multifunctional calmodulin-dependent protein kinase were distinct from several other "arginine-requiring" protein kinases.     

Purification of an insulin-sensitive cyclic AMP phosphodiesterase from rat liver

A low-Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola-cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE-substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 microM and a Vmax of 6.2 mumol mg-1 min-1 with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 microM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 microM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size-exclusion chromatography, suggesting that it exists as a dimer. A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin-activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin-sensitive phosphodiesterase.