Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A2 by acylamino phospholipid analogues.

The competitive inhibition of human pancreatic and a mutant human platelet phospholipase A2 (PLA2) was investigated using acylamino phospholipid analogues, which are potent competitive inhibitors of porcine pancreatic PLA2 [De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257]. Both the mutant platelet PLA2 and the human pancreatic PLA2 are effectively inhibited by these compounds. The enzyme from platelets is most strongly inhibited by compounds with a negatively charged phosphoglycol headgroup. Compounds with a neutral phosphocholine headgroup are only weak inhibitors, whereas an inhibitor with a phosphoethanolamine headgroup shows an intermediate inhibitory capacity. The platelet PLA2 is most effectively inhibited by negatively charged inhibitors having a relatively short (four or more carbon atoms) alkylchain on position one and a acylamino chain of 14 carbon atoms on position two. For the pancreatic enzyme an inhibitor with a phosphoethanolamine headgroup was more effective than inhibitors with either a phosphocholine or a phosphoglycol headgroup. The chainlength preference of the pancreatic enzyme resembles that of the platelet PLA2. The largest discrimination in inhibition between the human platelet and the human pancreatic PLA2 is obtained with inhibitors with a negatively charged phosphoglycol headgroup, an alkyl chain of four carbon atoms on position one and a long acylamino chain of 14-16 carbon atoms on position two. Because the platelet PLA2 is thought to have several biological functions, specific inhibitors of this enzyme could have important implications in the design of pharmaceutically interesting compounds.     

Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties

Residue 31 of porcine pancreatic phospholipase A2 (PLA2) is located at the entrance to the active site. To study the role of residue 31 in PLA2, six mutant enzymes were produced by site-directed mutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Ser or Gly. Direct binding studies indicated a three to six times greater affinity of the Trp31 PLA2 for both monomeric and micellar substrate analogs, relative to the wild-type enzyme. The other five mutants possess an unchanged affinity for monomers of the product analog n-decylphosphocholine and for micelles of the diacyl substrate analog rac-1,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine. The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholine were decreased 9-20 times for these five mutants. Kinetic studies with monomeric substrates showed that the mutants have Vmax values which range between 15 and 70% relative to the wild-type enzyme. The Vmax values for micelles of the zwitterionic substrate 1,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3-50 times. The Km values for the monomeric substrate and the Km values for the micellar substrate were hardly affected in the case of five of the six mutants, but were considerably decreased when Trp was present at position 31. The results of these investigations point to a versatile role for the residue at position 31: involvement in the binding and orientating of monomeric substrate (analogs), involvement in the binding of the enzyme to micellar substrate analogs and possibly involvement in shielding the active site from excess water.     

Cloning, expression, purification and characterization of patatin, a novel phospholipase A.

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.     

Comparison of two chemical cleavage methods for preparation of a truncated form of recombinant human insulin-like growth factor I from a secreted fusion protein

We have produced a naturally occurring variant of human insulin-like growth factor I, truncated by three amino acids at the amino terminus. The polypeptide is obtained as a fusion protein in Escherichia coli. The fusion partner is a synthetic IgG-binding peptide. During fermentation the fusion protein is secreted into the medium, and is purified on IgG--Sepharose prior to cleavage. Two different genes for the fusion protein were used, allowing chemical cleavage at either a tryptophan linker or a methionine linker between the fusion partner and the growth factor, using N-chlorosuccinimide (NCS) or cyanogen bromide (CNBr) respectively. A partial CNBr cleavage yielded the native peptide, whereas the NCS cleavage yielded a product in which the single methionine had been oxidized to the sulfoxide. The forms from both cleavage methods exhibited biological activity and were characterized after purification to homogeneity. Both cleavage methods gave products having correct N- and C-terminal ends. The purified product had a biological activity equal to that of corresponding material from natural sources, 15 000 U/mg. Modified forms of truncated IGF-I were also identified, purified and characterized. Modifications such as proteolysis and misincorporation of norleucine for methionine occurred during biosynthesis, while oxidation of methionine took place during both fermentation and chemical cleavage.     

Purification of a phospholipase A2 from human monocytic leukemic U937 cells. Calcium-dependent activation and membrane association

The existence of an intracellular phospholipase A2 (PLA2) involved in the production of 1-O-alkyl-sn-glycero-3-phosphocholine and free arachidonic acid has been repeatedly postulated. Using 1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as a substrate and a series of conventional and high-pressure liquid chromatographic techniques, we have purified a PLA2 from the soluble fraction of differentiated human monocytic U937 cells. The enzyme has been purified nearly 2000-fold to homogeneity. The purified enzyme has a molecular mass of 56 kDa, under reducing conditions, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity has a pH optimum of 8.0 and is calcium concentration-dependent. The EC50 for the activation of the enzyme activity by calcium is 300 nM. When the cells were homogenized in the presence of the calcium chelator EGTA (0.2 mM), the enzyme was found to be soluble (more than 90% of the activity in the 100,000 x g supernatant). However, when Ca2+ concentration was controlled from 10 nM to 100 microM in Ca2(+)-EGTA buffers, increasing amounts of the activity were found in the particulate fraction (100,000 x g pellet). This suggests that membrane translocation and activation of the soluble PLA2 may be regulated by physiological intracellular levels of Ca2+. The purified enzyme hydrolyzed different phosphatidylcholine substrates presented in either vesicular or Triton X-100 mix micellar forms. In both situations, the enzyme showed a high degree of specificity for arachidonic acid on the sn-2 position of the substrate. Substitution of palmitic or oleic on the sn-2 position substantially reduced the hydrolytic activity of the enzyme. When vesicles of arachidonic acid-containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were presented to the purified enzyme, all of them were hydrolyzed with comparable efficiency. However, only phosphatidylcholine and phosphatidylinositol were hydrolyzed when presented in Triton X-100 mixed micelles.     

Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.

Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain, M. K. (1991) Biochemistry 30, 7283-7297]. The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat. Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.     

Characterization of a glycosphingolipid beta-N-acetylgalactosaminyl-transferase activity in cultured hamster (nil) cells

The activity of a glycosphingolipid N-acetylgalactosaminyltransferase (GalNAc transferase) in cultured hamster fibroblasts (NIL-8) was characterized with respect to substrate binding, acceptor specificity, pH optimum and detergent requirements. Of the glycosphingolipid acceptors tested, transferase activity was observed only with globotriaosylceramide. The apparent Km values for uridinediphosphate-N-acetylgalactosamine and globotriasylceramide were 0.14 and 0.42 mM, respectively. The enzyme required Mn2+ for maximum activity (4 mM), and Mg2+ was not able to replace Mn2+. Of the detergents tested, sodium taurodeoxycholate gave the greatest activation of the enzyme at 1 mg/ml. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.0 in 2-(N-morpholino)ethanesulfonic acid. Globotetraosylceramide and II3-alpha-N-acetylneuraminyl-lactosylceramide inhibited transferase activity with globotriaosylceramide as substrate, but lactosylceramide had no effect on the activity with this acceptor. The major product of the assay was shown to be a tetraglycosylceramide with a terminal beta-N-acetylgalactosamine moiety by co-migration with authentic globotetraosylceramide on TLC plates and by cleavage of the labeled N-acetylgalactosamine from the product by jack bean beta-hexosaminidase.     

The primary structure of Lactobacillus casei thymidylate synthetase. I. The isolation of cyanogen bromide peptides 1 through 5 and the complete amino acid sequence of CNBr 1, 2, 3, and 5.

Thymidylate synthetase from Lactobacillus casei was S-carboxymethylated and degraded by treatment with cyanogen bromide. Although the protein contains 6 methionine residues, only 5 cyanogen bromide peptides were obtained due to the presence of 1 methionine on the NH2 terminus and another adjacent to a threonine residue which was resistant to cleavage. The peptides were isolated by differential extraction, first with ammonium acetate, then pyridine acetate, and finally the residue was solubilized with 50% acetic acid. Each peptide was further purified to homogeneity by Bio-Gel chromatography. The size of the peptides from the amino to carboxyl end of the enzyme subunit was CNBr 1, 4,100; CNBr 2, 10,300; CNBr 3, 8,100; CNBr 4, 11,800; CNBr 5, 2,200. The sum of the amino acid residues of the peptides is equal to the sum of the residues in an enzyme subunit, indicating that all of the CNBr peptides have been isolated. The CNBr-resistant methionine was located in CNBr 2 and the 5-fluoro-2'-deoxyuridine 5'-monophosphate binding site in CNBr 4. The holoenzyme molecular weight, based on the residue weights of the amino acids in the two equivalent subunits, is equal to 73,176. The complete sequence of each of the CNBr peptides, except for CNBr 4, which is presented in the following paper, is described.     

A simple assay for a human serum phospholipase A2 that is associated with high-density lipoproteins

Phospholipase A2 (PLA2) activity is usually assayed with expensive radioactive or chromogenic substrates unsuitable for performing large numbers of assays. We have designed a simple microplate assay for human serum PLA2 using the chromogenic substrate 4-nitro-3-octanoyloxy-benzoic acid. Using this substrate, serum PLA2 activity was similar to that measured with the previously characterized chromogenic phospholipid substrate 1,2-bis-heptanoylthio-glycerophosphocholine. However, the assay described here appears to be more sensitive. The mean PLA2 activity in serum from healthy volunteers (n = 30) measured by this assay was 10.4 +/- 1.6 micromol x h(-1) x ml(-1). The assay is reproducible and is suitable for the analysis of large numbers of samples in a clinical setting. We have also demonstrated that 94% of the PLA2 activity in normal human serum is associated with high-density lipoproteins and that serum PLA2 activity is positively correlated with the lipoprotein parameters total triglyceride (P < 0.0001), total cholesterol (P < 0.0001), and atherogenic index (P = 0.008). The serum PLA2 activity was calcium dependent and was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin (EC(50) = 0.4 mM). The PLA2 activity characterized here is unlikely to be due to plasma platelet-activating factor acetylhydrolase or low molecular weight His-Asp sPLA2, and may represent a new sPLA2 type.     

Kinetics of the hydrolysis of mixed micelles of dipalmitoyllecithin with triton X-100 catalyzed by a phospholipase A2 from the venom of Agkistrodon halys blomhoffii

The pH dependence of kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by the intact and the N-terminal alpha-NH2-modified phospholipases A2 (PLA2s) of Agkistrodon halys blomhoffii, was studied at 25 degrees C and ionic strength 0.1 in the presence of saturating amounts of Ca2+. The pH dependence of the kinetic parameters for the hydrolysis of monodispersed diC6PC, catalyzed by the modified enzyme, was also studied under the same conditions, and the data were compared with the previous results for the intact enzyme [Teshima, K. et al. (1986) J. Biochem. 100, 1655-1662]. The pK values of the catalytic group, His 48, and Tyr 52 were found to shift from 5.55 to 7.00 and from 10.50 to 11.50, respectively, on binding of the micellar substrates to the enzyme. On the other hand, no participation of these ionizable groups was observed for the binding of the monodispersed substrate. On the basis of the present finding and the X-ray crystallographic studies on bovine pancreatic PLA2 [Dijkstra, B.W. et. al. (1981) J. Mol. Biol. 147, 97-123] and on a PLA2 of Crotalus atrox venom [Brunie, S. et al. (1985) J. Biol. Chem. 260, 9742-9749], the hydrogen-bonding of Tyr 73, which is involved in the lipid-water interface recognition site, to His 48 and Tyr 52 in the active center was strongly suggested to be important for the hydrolysis of micellar substrates.     

The behavior of proteases in lecithin reverse micelles

Reverse micelles, formed in isooctane/alcohol by phosphatidylcholines of variable chain length (i.e. 6, 7 or 8 C atoms in the fatty acid moiety) have been studied, mostly in relation to their capability of solubilizing trypsin and alpha-chymotrypsin. It has been found that the capability of the lecithin reverse micellar systems to solubilize water is strongly affected by the chain length of the alkyl group and by the alcohol used as co-surfactant. The C8-lecithin system, i.e. 1,2-dioctanoyl-sn-glycero-3-phosphocholine, in isooctane/hexanol is the system which affords the maximal solubilization of water (up to wo 60, where wo = [H2O]/[lecithin]) and of the enzymes. The water of the water pool of lecithin reverse micelles has been investigated by 1H-NMR; the proton chemical shift as a function of wo was found to be similar to the case of reverse micelles formed by the well known negatively charged surfactant sodium bis(2-ethylhexyl sulfosuccinate). 31P-NMR studies show that the ionization behavior of phosphate groups is similar to that in bulk water, suggesting no anomaly in the pH behavior of this water pool. The stability of trypsin and alpha-chymotrypsin in the various lecithin reverse micellar system is similar and occasionally better than that in aqueous solution. The same holds for the kinetic behavior (kcat and Km have been determined for a few systems). The bell-shaped curve of the pH/activity profile in lecithin reverse micelles is, for both enzymes, shifted towards more alkaline values with respect to water. Bell-shaped curves are also obtained when studying the influence of wo on the enzyme activity, with an optimal wo which is in the range 7-10, a surprisingly small value considering that we are dealing with hydrolases. Circular dichroic studies have been carried out in order to correlate the activity with the protein conformation: for both enzymes, generally no marked perturbations appear as a consequence of the solubilization in the lecithin reverse micelles, but conditions can be found under which significant alterations are present. Certain properties of the two enzymes, which in water solution are very similar, become sharply different in reverse micelles, showing that occasionally the micellization is able to enhance the relatively small structural differences between the two proteins.     

Use of short-chain cyclopentano-phosphatidylcholines to probe the mode of activation of phospholipase A2 from bovine pancreas and bee venom

A great mystery in the mechanism of phospholipase A2 (PLA2) and many other lipolytic enzymes is the "interfacial activation" induced by micellar but not monomeric substrates. Equally mysterious is the lack of interfacial activation in bee venom PLA2, as opposed to PLA2s from pancreas and other sources. We have probed these problems using the conformationally restricted short-chain cyclopentano-analogues of diacylphosphatidylcholine (Cp-DCnPC, all-trans isomer). In the reaction catalyzed by bovine pancreatic PLA2, Cp-DC8PC behaved differently from DC8PC in that its monomers and micelles showed comparable activities (but lower than the activity of DC8PC). This result suggests that the activity of PLA2 can be regulated by substrate conformation and supports the "substrate conformation model" (Wells, M. A. (1974) Biochemistry 13, 2248-2257), but raises a question as to whether Cp-DC8PC mimics monomers or micelles of DC8PC. Conformational analysis by 1H NMR revealed that monomeric Cp-DC8PC was conformationally restricted near the carbonyl region, a property characteristic of micelles. Thus, monomeric CP-DC8PC can be considered as a conformational analogue of micelles, but the important structural feature lies in the CH2COO region instead of the glycerol backbone. CP-DC8PC was then used to test a previous proposal that the bee venom PLA2 hydrolyzes monomers but not micelles (which would predict little or no activity for Cp-DC8PC since its conformation is micelle-like whether below or above its critical micelle concentration). The results showed that Cp-DC8PC is a relatively good substrate for the bee venom PLA2 in comparison with the pancreatic PLA2. This and other evidence together suggest that the bee venom PLA2 is not sensitive to the conformation of monomeric and micellar substrates and hydrolyzes both monomers and micelles. The results in both PLA2s demonstrate the usefulness of cyclopentano-phospholipids in probing the mechanism of phospholipases and the roles of substrate conformation in the catalysis of PLA2.     

Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.

Studies of the specificity of phospholipases A2 (PLA2s) for different substrates have usually been carried out in vesicles or mixed micelles, where differences in shape, size, or charge of vesicles formed with different phospholipids may give misleading results. Another factor is binding of the enzyme to the phospholipid surface, which has recently been addressed using vesicles of an anionic phospholipid, dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) to which some extracellular PLA2s were shown to bind with a very high affinity (Jain, M. K., and Berg, O. G. (1989) Biochem. Biophys. Acta 1002, 127-156). In the present report we have used a similar system to study the substrate preferences of two human PLA2s that are thought to be physiologically relevant in the metabolism of arachidonic acid: a recombinant form of the human synovial fluid (14 kDa) PLA2 and the cytosolic (85 kDa) PLA2 found in monocytic cells. It is shown that both human enzymes bind tightly to DMPM vesicles and follow the basic characteristics of processive hydrolysis in this model using analysis of progress curves and substrate competition experiments. Mixed vesicles containing DMPM with small amounts (3-5 mol%) of other phospholipids have been used to study the substrate selectivity of the two human isoenzymes. The synovial fluid PLA2 shows a clear preference (approximately 7-fold) for sn-glycero-3-phosphoethanolamine over sn-glycero-3-phosphocholine. Within glycerophosphocholines, this enzyme displays little preference for the sn-2 fatty acyl group, and a slight preference for phospholipids with sn-1-acyl versus sn-1-alkyl substituents. In contrast, the cytosolic PLA2 shows a marked selectivity for arachidonoyl in the sn-2 position and only minor differences in selectivity for the polar head group in the sn-3 position. This enzyme does not distinguish between sn-1-acyl and sn-1-alkyl subclasses of glycerophosphocholines.     

Evolution of a rippled membrane during phospholipase A2 hydrolysis studied by time-resolved AFM

The sensitivity of phospholipase A(2) (PLA(2)) for lipid membrane curvature is explored by monitoring, through time-resolved atomic force microscopy, the hydrolysis of supported double bilayers in the ripple phase. The ripple phase presents a corrugated morphology. PLA(2) is shown to have higher activity toward the ripple phase compared to the gel phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes, indicating its preference for this highly curved membrane morphology. Hydrolysis of the stable and metastable ripple structures is monitored for equimolar DMPC/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-supported double bilayers. As shown by high-performance liquid chromatography results, DSPC is resistant to hydrolysis at this temperature, resulting in a more gradual hydrolysis of the surface that leads to a change in membrane morphology without loss of membrane integrity. This is reflected in an increase in ripple spacing, followed by a sudden flattening of the lipid membrane during hydrolysis. Hydrolysis of the ripple phase results in anisotropic holes running parallel to the ripples, suggesting that the ripple phase has strip regions of higher sensitivity to enzymatic attack. Bulk high-performance liquid chromatography measurements indicate that PLA(2) preferentially hydrolyzes DMPC in the DMPC/DSPC ripples. We suggest that this leads to the formation of a flat gel-phase lipid membrane due to enrichment in DSPC. The results point to the ability of PLA(2) for inducing a compositional phase transition in multicomponent membranes through preferential hydrolysis while preserving membrane integrity.     

Proteolytic sensitivity of a recombinant phospholipase D from cabbage: identification of loop regions and conformational changes

A recombinant phospholipase D from white cabbage (PLD2) composed of 812 amino acid residues was studied by site-directed mutagenesis and limited proteolysis to obtain first information on its tertiary structure. Limited proteolysis by thermolysin resulted in the formation of some large fragments of PLD2. From mass spectrometry and N-terminal sequencing of the peptides, the cleavage sites could be identified (1. Thr41-Ile42, 2. Asn323-Leu324 or Gly287-Leu288 and Ser319-Ile320 in case of the mutant L324S-PLD2). This suggested an exposed loop in the C2 domain of PLD2 and a large flexible region close to the N-terminal side of the first catalytic (HKD) motif. Calcium ions, the substrate 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and the competitive inhibitor 1,3-dipalmitoylglycero-2-phosphocholine influenced the proteolytic cleavage. Calcium ions exerted a destabilizing effect on the conformation of PLD2.     

Phosphorylation of heart glycogen synthase by cAMP-dependent protein kinase. Regulatory effects of ATP

Glycogen synthase I, purified from bovine heart, had a specific activity of 33 units/mg and gave a single band on sodium dodecyl sulfate gel electrophoresis with a subunit molecular weight of 86,000. The enzyme was phosphorylated with cAMP-dependent protein kinase catalytic subunit, also isolated from heart. With 10 microM ATP, only one phosphate group was incorporated per subunit of glycogen synthase. The phosphorylation decreased the per cent of glycogen synthase I from 0.95 to 0.50 when activity was determined by assays with Na2SO4 and glucose 6-phosphate. Glycogen synthase containing one phosphate per subunit was designated GS-1. One additional phosphate was incorporated per synthase subunit when ATP was increased to 0.5 mM and the percent glycogen synthase I decreased from 0.50 to < 0.05. This enzyme form was designated GS-1,2. Conversion of GS-1 to Gs-1,2 gave cooperative kinetics with ATP concentration and a half-maximal stimulation at approximately 40 microM. Phosphorylation of GS-1 could also be achieved by adding other non-substrate nucleotide triphosphates such as ITP and UTP along with 10 microM ATP. Glucose-6-P and Na2SO4 were without effect on this phosphorylation reaction. Two separate peptides were obtained after CNBr cleavage of 32P-labeled GS-1,2 and only one from GS-1. Both enzyme forms contained a single phosphorylated peptide in common. Thus, heart glycogen synthase may be phosphorylated specifically in either of two different sites using appropriate concentrations of ATP. ATP acts as a substrate for the protein kinase and also affects the availability of a second site to phosphorylation by cAMP-dependent protein kinase.     

Chiral synthesis of a dithiolester analog of phosphatidylcholine as a substrate for the assay of phospholipase A2

The synthesis of a dithiolester analog of phosphatidylcholine, 1,2-bis(heptanoylthio)-1,2-dideoxy-sn-glycerol-3-phosphocholine (thio PC), is described. Starting with 1-trityl-sn-glycerol (prepared from D-mannitol), tosylation followed by displacement with potassium methyl xanthate gave a trithiocarbonate. Reductive cleavage of the latter gave a 1,2-dithiol which was then acylated, detritylated, and esterified with choline phosphate. Hydrolysis of thio PC by phospholipase A2 (Naja naja) indicated 95% chiral purity. The rate of hydrolysis as a function of substrate concentration showed a sharp increase at about 0.17 mM, the critical micellar concentration of the lipid. A spectrophotometric assay of phospholipase A2 (by measurement of released thiol groups in the presence of dithionitrobenzoic acid) was quite sensitive. As little as 1 ng of enzyme was detected, representing a rate of about 0.2 nmol of substrate hydrolyzed per min.     

Micelle-vesicle transition in phospholipid-bile salt mixtures. A study by precision scanning calorimetry

A systematic study of the micelle-vesicle transformation in phospholipid-bile salt mixtures using differential scanning calorimetry (DSC) indicates that the lipid undergoes a variety of changes in its thermal properties as mixed micellar solutions are diluted to concentrations at which vesicles form. In the experiments, micellar solutions of 50 mg/mL total lipid, containing sodium taurocholate (TC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), are diluted to concentrations corresponding to differing extents of aggregation of the TC with phospholipid. Turbidity and equilibrium dialysis measurements are used to establish boundaries between where micelles persist and where vesicles are formed and to determine the extent of aggregation of the TC with DPPC. At molar ratios Re of bound TC to DPPC greater than 0.3, micellar solutions are formed, while at Re less than 0.15 vesicles are evident upon dilution. As the transformation from micelles to vesicles occurs, the thermal transitions in the lipid change from broad, low Cp (max) peaks in the micelle region to multiple peaks of high cooperativity in regions of composition where lamellar structures and vesicles form. The DSC curves show that in the composition region corresponding to where bilayer micelles exist a new thermal phase forms, which has a melting transition near 32 degrees C, if the solutions are allowed to equilibrate for 48 h at 21 degrees C. Furthermore, at compositions between Re = 0 and 0.25, there is metastability in the lipid when equilibrated at 21 degrees C, but heating the lipid through the thermal transitions leads to reversible behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins

Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.     

Degradation of n-haloalkanes and alpha, omega-dihaloalkanes by wild-type and mutants of Acinetobacter sp. strain GJ70.

A 1,6-dichlorohexane-degrading strain of Acinetobacter sp. was isolated from activated sludge. The organism could grow with and quantitatively release halide from 1,6-dichlorohexane, 1,9-dichlorononane, 1-chloropentane, 1-chlorobutane, 1-bromopentane, ethylbromide, and 1-iodopropane. Crude extracts contained an inducible novel dehalogenase that liberated halide from the above compounds and also from 1,3-dichloropropane, 1,2-dibromoethane, and 2-bromoethanol. The latter two compounds were toxic suicide substrates for the organism at concentrations of 10 and 5 microM, respectively. Mutants resistant to 1,2-dibromoethane (3 mM) lacked dehalogenase activity and did not utilize haloalkanes for growth. Mutants resistant to both 1,2-dibromoethane (3 mM) and 2-bromoethanol (30 mM) could no longer oxidize or utilize alcohols and were capable of hydrolytic dehalogenation of 1,2-dibromoethane to ethylene glycol.