Enzyme-phospholipid Complex Research Articles

Benzene-di- N-octylcarbamates as conformationally constrained phospholipase A 2 inhibitors

Conformationally constrained 1,2-, 1,3-, and 1,4-benzene-di- N-octylcarbamates are potent reversible competitive inhibitors of Naja mocambique mocambique phospholipase A 2 with the K i values of 11, 4, and 15 μM, respectively. With the angle of 120 o between two C(benzene)–O bonds, 1,3-benzene-di- N-octylcarbamate mimics the preferable eclipsed C( sn-2)–O/C( sn-3)–O conformer of phospholipid in the enzyme-phospholipid complex. Further, a three-step phospholipase A 2 inhibition mechanism by the inhibitor is proposed.

Mycobacterium smegmatis malate dehydrogenase: activation of the lipid-depleted enzyme by anionic phospholipids and phosphatidylethanolamine

Phospholipid-protein interactions have been investigated in a phospholipid-requiring enzyme, FAD-dependent malate dehydrogenase isolated from Mycobacterium smegmatis membranes, to correlate these interactions with enzyme function. The ability of several natural and synthetic phospholipids including CL and PE, which are major phospholipids in M. smegmatis membranes, to activate purified, lipid-depleted, enzymatically inactive malate dehydrogenase was examined. Anionic phospholipids and PE activated the enzyme, while zwitterionic phospholipids did not. A PE/PC mixture activated the enzyme in the form of both bilayer and non-bilayer structure. CL/PE mixtures activated malate dehydrogenase much more than each single phospholipid species. All anionic phospholipids used stabilized the enzyme, while PE and zwitterionic phospholipids did not. CL and a CL/PE mixture protected malate dehydrogenase from proteinase digestion, while PE did not. All phospholipids and phospholipid mixtures tested caused little secondary structural change in malate dehydrogenase. The results obtained in this study suggest that CL and CL/PE mixtures could form stable, enzymatically active complexes with malate dehydrogenase which might be similar to the native complex in M. smegmatis membranes. Although PE could activate malate dehydrogenase in both bilayer and non-bilayer form, it formed a complex with malate dehydrogenase which was inferior in terms of stability and susceptibility to proteinases, indicating that PE alone poorly reconstitutes the active enzyme-phospholipid complex.

In vitro alterations of L-asparaginase activity of Tetrahymena pyriformis by lipids.

A membrane-bound L-asparaginase (EC 3.5.1.1) of Tetrahymena pyriformis was purified to homogeneity. The purified enzyme is a lipoprotein, since it is inactivated by phospholipase C and its activity is restored by the addition of naturally occurring lipids, such as phosphatidylcholine, triolein and oleyl acetate. The relative effectiveness of a variety of phospholipids, free saturated and unsaturated fatty acids, or neutral lipids, such as esters of fatty acids and glycerides, with respect to the activation of purified L-asparaginase is compared. Enzyme activity is reconstituted in the presence of lipids and evidence for the formation of an enzyme-phospholipid complex is presented. The data of this report suggest that L-asparaginase may have a requirement for lipids that reconstitute a physiological hydrophobic environment, similar to the one existing in vivo.

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside.

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.

Phospholipid protection against proteolysis of D-beta-hydroxybutyrate dehydrogenase, a lecithin-requiring enzyme.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly inactivates D-beta-hydroxybutyrate dehydrogenase; the addition of NAD+ or NADH, together with dithiothreitol and increased salt (to 50 mM), decreases the rate of inactivation, and with trypsin, virtually eliminates inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversible modification of D-beta-hydroxybutyrate dehydrogenase by diamide.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for function. The purified enzyme devoid of lipid (apodehydrogenase) is inactive but can be reactivated by forming a complex with phospholipid containing lecithin. We find that, of the six half cysteines present in D-beta-hydroxybutyrate dehydrogenase, only two are in the reduced form and available for modification with N-ethylmaleimide, even after denaturation in sodium dodecyl sulfate. Diamide treatment of either the inactive apodehydrogenase or the active enzyme-phospholipid complex resulted in complete loss of enzymic activity, the apodehydrogenase being assayed after addition of phospholipid. The inactivation by diamide can be reversed by the addition of dithiothreitol with full recovery of activity. Derivatization using N-[14C]ethylmaleimide showed that diamide modified only one sulfhydryl per enzyme monomer. The other sulfhydryl appears not to be essential for function since full activity can be restored after this sulfhydryl had been covalently derivatized with N-ethylmaleimide. Protein cross-linking was not observed after diamide modification of D-beta-hydroxybutyrate dehydrogenase, indicating that a disulfide bridge was not formed between enzyme subunits. The diamide-modified enzyme retains the ability to bind coenzyme, NAD(H), as detected by quenching of the intrinsic fluorescence of the protein. However, resonance energy transfer from protein to bound NADH and enhancement of NADH fluorescence were not observed, indicating that diamide modification of the protein alters the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Half-site reactivity of an essential thiol group of D-beta-hydroxybutyrate dehydrogenase.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme, which is a tetramer both in the mitochondrial inner membrane and as the purified enzyme reconstituted with phospholipid. For the active enzyme-phospholipid complex in the absence of ligands, we previously found that reaction with N-ethylmaleimide (at 5 mol/mol of enzyme subunit) resulted in progressive loss of enzymic activity with an inactivation stoichiometry of 1 equiv of sulfhydryl derivatized per mole of enzyme and a maximum derivatization of 2 equiv [Latruffe, N., Brenner, S. C., & Fleischer, S. (1980) Biochemistry 19, 5285-5290]. We now find, in the presence of nucleotide or substrate, that the rate of inactivation is significantly reduced, which indicates that these ligands afford protection of the essential sulfhydryl. Further, in the presence of ligands, the inactivation stoichiometry is 0.5, consistent with half-of-the-site reactivity of the essential sulfhydryl. Thus, at a low ratio of N-ethylmaleimide to enzyme, nucleotide or substrate affords essentially complete protection of the nonessential sulfhydryl from derivatization. The binding characteristics of NADH to both the native and N-ethylmaleimide-derivatized enzyme have been compared by fluorescence spectroscopy. Quenching of intrinsic tryptophan fluorescence of the protein shows that the enzyme, derivatized with N-ethylmaleimide either in the absence or in the presence of NAD+, binds NADH but with a reduced Kd (approximately 50 microM as compared with approximately 20 microM for native enzyme). However, a critical change has occurred in that resonance energy transfer from protein to bound NADH, observed in the native enzyme, is abolished in the N-ethylmaleimide-derivatized enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of arginines in d-β-hydroxybutyrate dehydrogenase

d-β-Hydroxybutyrate dehydrogenase ( d-3-hydroxybutyrate: NAD + oxidoreductase, EC 1.1.1.30) is a lipid-dependent enzyme which has an absolute and specific rquirement for phosphatidylcholine for function. Chemical derivatization studies using 1,2-cyclohexanedione, an arginine-specific reagent, have been carried out on the purified enzyme devoid of lipid as well as on the enzyme reactivated with phospholipid. Cyclohexanedione inactivated the active enzyme-phospholipid complex and the lipid-free enzyme was rendered inactivatable by phospholipid. From kinetic studies and by direct chemical derivatization studies with [1- 14C]cyclohexanedione, we find that incorporation of a single cyclohexanedione molecule per enzyme monomer resulted in complete loss of enzymic activity. The presence of NADH or NAD +, cofactors for the enzyme, offered no protection for the rate of inactivation. The substrates β-hydroxybutyrate and acetoacetate with or without coenzyme gave little or no protection. However, 2-methyl malonate, a competitive inhibitor for β-hydroxybutyrate, strongly protected against inactivation. These studies indicate that: (1) a single arginine serves a vital role and is essential for function; (2) the arginine is located in the proximity of the substrate binding site.

Differential effects of phospholipids on skeletal alkaline phosphatase activity in extracts, in situ and in circulation

Human skeletal alkaline phosphatase (ALP) purified from human bone was subject to competitive inhibitions by phospholipids including cephalins, lecithins, and phosphatidylinositol. K i values ranged from 0.7 to 1.5 m m, at pH 9.5. As previously shown, the enzyme was subject to uncompetitive inhibition by imidazole. The inhibitory phospholipids potentiated this effect, and altered the nature of the imidazole inhibition, from uncompetitive to mixed type, suggesting that imidazole was bound more efficiently to the enzyme-phospholipid complex than to the enzyme-substrate complex. No interactions were observed between phospholipids and other uncompetitive inhibitors of ALP. The skeletal ALP activity of cultured chick calvarial cells was assayed both in situ and in extracts. Like the extracted human isoenzyme, the extracted chick ALP was subject to competitive inhibition by cephalin ( K i = 0.3 mM at pH 9.3) and an inhibitory interaction between cephalin and imidazole, but the same isoenzyme showed neither effect in situ. The value of K m,PNPP at pH 9.5 for chick skeletal ALP was 1.5 m m in extracts and 7.1 m m in situ. When embryonic chick bones were cultured in vitro, skeletal ALP activity was released into the serum-free medium. Unlike the same isoenzyme extracted from the bones, the ALP activity in the medium was not inhibited by cephalin and showed no inhibitory interaction between cephalin and imidazole. Similarly, human serum ALP activities were not as sensitive to phospholipid inhibition as the same isoenzymes extracted from tissues. Human skeletal ALP extracted from bone was inhibited by cephalin, but the skeletal isoenzyme in Pagetic serum was not, suggesting that the potential for phospholipid interaction was altered during or after release from osteoblast cell membranes. The observation that extracted human skeletal ALP lost its potential for inhibition by phospholipids after treatment with phospholipase C further suggests that ALP activity may be released from cells during membrane turnover.

Target size of D-beta-hydroxybutyrate dehydrogenase. Functional and structural molecular weight based on radiation inactivation.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apoenzyme has been purified to homogeneity from beef heart; it is devoid of lipid and inactive. It can be functionally reconstituted with lecithin or phospholipid mixtures containing lecithin. The active form of the enzyme is the enzyme-phospholipid complex. Classical target analysis of radiation-inactivation data has now been used to determine the molecular size of the enzyme both in the native membrane (submitochondrial vesicles) and in the reconstituted enzyme inserted into phospholipid vesicles containing lecithin. For both forms of the enzyme, we find the same molecular size, approximately 110,00 daltons. This size is consistent with a tetramer. Radiation results in fragmentation of the polypeptide and the destruction of the polypeptide correlates with loss of enzymic function. A similar size is obtained when purified D-beta-hydroxybutyrate dehydrogenase is inserted into a nonactivating mixture of phospholipid (i.e. in the absence of lecithin). We conclude that: 1) the native enzyme in submitochondrial vesicles and the purified active enzyme in phospholipid vesicles are the same size, approximating a tetramer; 2) radiation of D-beta-hydroxybutyrate dehydrogenase results in loss of activity and fragmentation of the polypeptide; and 3) the role of lecithin in activation of D-beta-hydroxybutyrate dehydrogenase is unrelated to determining oligomeric size of the enzymes since both active and nonactive forms exhibit the same structural size.

The biosynthesis of oligosaccharide-lipids. Activation of mannosyltransferase II by specific phospholipids.

Mannosyltransferase II catalyzes transfer directly from GDP-mannose to an oligosaccharide-lipid resulting in the formation of the alpha-1,3-mannosyl linkage in Man alpha 1-3(Man alpha 1-6)Man beta-GlcNAc beta-GlcNac-P-P-lipid. The enzyme has been solubilized and purified 660-fold from rabbit liver microsomes (Jensen, J. W., and Schutzbach, J. S. (1981) J. Biol. Chem. 256, 12899-12904). Enzyme activity was reconstituted in the presence of specific phospholipids and evidence for the formation of an enzyme-phospholipid complex was obtained from kinetic studies, by the stabilization of the enzyme by phosphatidylethanolamine, and by co-sedimentation of the enzyme with phospholipid. Maximal activity was restored only when the enzyme was reconstituted in the presence of synthetic or naturally occurring phosphatidylethanolamine which contained unsaturated acyl chains. Investigation of the phospholipid specificity required for activation of the enzyme and results obtained with phospholipid vesicles of mixed composition suggest that the enzyme may have a requirement for phospholipids that can associate to form nonbilayer lipid structures in aqueous environments.

Transmembrane electron transfer in an enzyme-phospholipid complex

Purified malate-vitamin K reductase (MKR) from Mycobacterium phlei , which requires phospholipid and FAD for the enzymatic reduction of dye electron acceptors, was used as an electron transfer system in combination with synthetic phospholipid vesicles. Ferricyanide entrapped inside the sonically prepared vesicles was enzymatically reduced by MKR. The rate of reduction of ferricyanide by MKR was dependent upon a lipid soluble mediator. The rate of enzymatic reduction of ferricyanide by malate-vitamin K reductase was increased by the addition of phenazine methosulfate (PMS) or benzoquinone.

A Phospholipid-requiring Enzyme, Malate-Vitamin K Reductase: PURIFICATION AND CHARACTERIZATION

Abstract Malate-vitamin K reductase has been purified to near homogeneity from Mycobacterium phlei. The purified enzyme is dependent upon an added phospholipid for activity and requires FAD as a cofactor. The enzymatic activity was found to be affected by the degree of enzyme aggregation. At high salt concentrations the enzyme existed in a monomeric form which was more active than the aggregated form. The enzyme was reversibly aggregated into a less active form by either dilution or dialysis against a buffer of low salt concentration. An enzyme-phospholipid complex was isolated by glycerol gradient centrifugation. It is suggested that a phospholipid binding site (or sites) seems to be involved in the aggregation-disaggregation process. The molecular weight of the monomeric form was determined to be 53,000 by Sephadex G-200 chromatography and 51,000 by sodium dodecyl sulfate gel electrophoresis, whereas the aggregated form had a molecular weight of approximately 164,000, as estimated by Sephadex G-200.

Isolation and Purification of a 5α-Hydroxysterol Dehydrase of Yeast

Abstract An enzyme that catalyzes the anaerobic dehydration of ergosta-7,22-diene-3β,5α-diol to ergosterol has been isolated from baker's yeast (Saccharomyces cerevisiae). The enzyme has been purified about 500-fold by ammonium sulfate precipitation, chromatography on Sephadex G-100, and adsorption onto calcium phosphate gel. No cofactors are required for activity; 2-mercaptoethanol was added to all buffers. Throughout purification, phospholipid and ergosterol are associated with the active enzymic fraction. The lipid may be removed by digestion with phospholipase A; gross changes in the physical appearance of the purified enzyme-phospholipid complex are observed following phospholipid digestion. However, the phospholipid may be completely digested without significant loss of dehydrase activity. Many assays of the dehydrase activity have been developed. In two spectrophotometric assays, ergosterol formation is measured by absorbance at 282 nm, either directly in the reaction mixture or indirectly following saponification and extraction. Substrate disappearance and product formation have been measured both by quantitative gas-liquid chromatography and by conversion of 14C-labeled substrate into ergosterol. The rates of formation of ergosterol measured by each assay method are identical. The disappearance of substrate and the appearance of ergosterol are equimolar.