Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.

Presence of glycerol masks the effects of phosphorylation on the catalytic efficiency of cytosolic phospholipase A2.

Cytosolic phospholipase A2 catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. The enzymatic activity of cPLA2 is affected by several mechanisms, including substrate presentation and the phosphorylation state of the enzyme. Using covesicles of 1-palmitoy1-2-arachidonoyl-[arachidonoyl-1-14C]-8n-glycero-3 -phosphocholine and 1,2-dimyristoyl-phosphatidylmethanol as substrate, the effects of phosphorylation on the interfacial binding and catalytic constants were investigated. Phosphorylated and dephosphorylated enzyme forms were shown to have identical values of 2.6 microM for KMapp, an equilibrium dissociation constant which consists of the intrinsic dissociation constant from the lipid/water interface (Ks) and the dissociation constant for phospholipid from the active site (KM*). Moreover, the values of KM* for phosphorylated and dephosphorylated enzyme did not differ significantly (0.4 +/- 0.1 and 0.2 +/- 0.1, respectively). However, dephosphorylation of the enzyme reduced the value of kcat by 39%. The phosphorylation state of the enzyme had no effect on either the cooperativity shown by this enzyme or the thermal stability of the enzyme. Surprisingly, the presence of glycerol (4 M) masks the effect of phosphorylation on kcat. Instead, glycerol increased the value of kcat by 440% for the phosphorylated enzyme and by 760% for the dephosphorylated form. Moreover, addition of glycerol had only small effects on KMapp. the increase in the kcat upon addition of glycerol results from a substantial decrease in the activation energy from 29.4 to 14.8 kcal. mol-1. To determine whether the effects of phosphorylation of the enzyme or addition of glycerol are unique to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With these membranes, the dephosphorylated enzyme was only 21% less active than the phosphorylated enzyme. In the presence of glycerol, there was no detectable difference the two enzyme forms, and the rate of hydrolysis was increased by 300-390% over that measured in the absence of glycerol. These results suggest that the catalytic efficiency of the phosphorylated enzyme is not particularly relevant to its activation in vivo. Moreover, it may be that glycerol is mimicking the effect of some unidentified factor which greatly enhances the catalytic efficiency of the enzyme.

Cooperativity and binding in the mechanism of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phosphoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fusion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine (PAPE) does not show the same cooperative effect, although the rate at which it is hydrolyzed is much greater when PAPC is present. Moreover, PAPE has a dissociation constant from the active site (KD* = 0.7 mol %) which is comparable to that of PAPC and SAPI (KD* values of 0.3 and 0.3 mol %, respectively). These results are consistent with the presence of an allosteric site that, when occupied, induces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allosteric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilayer concentration of arachidonate-containing phospholipids. This previously unobserved effect may represent another mechanism by which cells can regulate the activity of cPLA2.

Active recombinant human cytosolic phospholipase A2 is expressed in Escherichia coli.

The cDNA encoding human cytosolic phospholipase A2 (cPLA2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme. Soluble, active fusion protein, designated His-cPLA2, has been obtained reproducibly from this expression system using the E. coli strain BL21 (DE3). The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry. His-cPLA2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA2 in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles. Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition metal ions. Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters. This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA2 and is advantageous for rapid production of protein and mutational analyses.

Role of histidine 373 in the catalytic activity of coagulation factor XIII.

Factor XIII catalysis proceeds via formation of thioester acyl enzyme intermediate involving an active site cysteine residue at position 314. The contribution of other residues to catalysis has not been established. Earlier studies of the pH dependence of factor XIII activity suggested the existence of a putative active site histidine. We used chemical modification and oligonucleotide directed site-specific mutagenesis to investigate the role of histidines. Photo-oxidation with methylene blue resulted in a complete loss of catalytic activity under conditions that oxidized histidine but did not affect the essential cysteine. Single substitution of each of the 14 histidine residues in the a-subunit of factor XIII by asparagine or alanine led to mutants with catalytic activities generally not significantly different from the wild-type recombinant enzyme. The only exceptions were the H373N and H373A mutants that were poorly expressed, had no detectable rate of [14C]putrescine incorporation into dimethylcasein, and failed to cross-link fibrin gamma-chains. Thus, the a-subunit His-373 may function in the active site of factor XIII, by analogy with papain's mechanism, as a histidinium cation that increases the nucleophilicity of the essential Cys-314. Decreased expression levels of His-373 mutants also indicate that this residue may be critical for enzyme stability.

Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide.

Many nascent proteins that are destined to be anchored to plasma membranes by a phosphatidylinositol glycan (PI-G) are in the range of 50-70 kDa so that changes of 2-3 kDa between precursors and products during processing are not easily detected. Furthermore, PI-G-anchored proteins are generally glycosylated so that changes between the nascent (prepro) proteins and the mature products are not due simply to the loss of signal peptides. These problems have made it difficult to monitor the processing of the prepro form of wild type human placental alkaline phosphatase (PLAP) in a cell-free system. We have designed a smaller and simpler substrate of PI-G "transamidase" derived by deletion of approximately 60% of the internal sequence of preproPLAP 513. This engineered protein, preprominiPLAP 208, retains the NH2- and COOH-terminal signal peptides of PLAP as well as all the epitopes for site-directed antibodies of the latter, but is devoid of glycosylation sites, the active site, and most of the cysteine residues. With preprominiPLAP, it has been possible to demonstrate, in a cell-free system, step by step conversion to the pro form and then to the mature form, with the concomitant loss of the appropriate signal peptides. These changes were shown to be time- and enzyme concentration-dependent. Studies with Asp-179 site-directed mutants of preprominiPLAP showed the same specificity for amino acids with a monosubstituted beta carbon at the cleavage/attachment site that were found previously with wild type PLAP.

Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined.

Nascent precursors of phosphatidylinositol-glycan (PI-G)-linked membrane proteins contain a hydrophobic COOH-terminal sequence of 15-30 residues that is eliminated during processing to yield a newly exposed COOH terminus to which the PI-G moiety is added. There is no consensus as to the primary structure of the terminal peptide but there is a specific requirement for the amino acid destined to become the COOH terminus. In nascent human placental alkaline phosphatase (PLAP), the PI-G tail is attached to Asp-484. Site-directed mutants with glycine, alanine, cysteine, serine, or asparagine (category I) at residue 484 become PI-G tailed, appear in the plasma membrane, and are enzymatically active when expressed in COS cells. Although mutants with glutamic acid, glutamine, proline, tryptophan, leucine, valine, phenylalanine, threonine, methionine, and tyrosine (category II) are expressed equally well, only small amounts appear on the plasma membrane. Furthermore, they are not PI-G tailed and have little alkaline phosphatase activity. Studies with truncated PLAP-489 rule out nonspecific conformational changes in category II mutant proteins as a reason for their failure to be processed in COS cells and point to a specific COOH-terminal processing enzyme. Direct evidence that the selectivity for category I amino acids is enzymatically determined was obtained in a cell-free translation/processing system by using rabbit reticulocyte lysate and CHO cell rough microsomal membranes. In this in vitro system, both category I and category II mutants of PLAP-513 were translated, glycosylated, and cleaved by NH2-terminal signal peptidase. However, an additional and selective cleavage at residue 484 was observed only with category I mutants.

Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte.

The Xenopus oocyte is widely used to study the various aspects of eukaryotic cell structure and function. It is also being used increasingly in expression cloning of cDNAs encoding proteins for which there are no structural data. One of the drawbacks of the Xenopus oocyte system is that individual oocytes taken at the same time from the same frog vary considerably in the amount of protein synthesized from the same amount of injected mRNA. In this report we describe the preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily determined in the medium of cultured oocytes by using a standard colorimetric assay. The amounts of alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes.

Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase.

Many proteins are now known to be anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety that is attached to their COOH termini. Placental alkaline phosphatase (PLAP) has been used as a model for investigating mechanisms involved in the COOH-terminal processing of PI-G-tailed proteins. The COOH-terminal domain of pre-pro-PLAP provides a signal for processing during which a largely hydrophobic 29-residue COOH-terminal peptide is removed, and the PI-G moiety is added to the newly exposed Asp-484 terminus. This cleavage/attachment site was subjected to an almost saturation mutagenesis, and the enzymatic activities, COOH-terminal processing, and cellular localizations of the various mutant PLAP forms were determined. Substitution of Asp-484 by glycine, alanine, cysteine, asparagine, or serine (category I) resulted in PI-G-tailed and enzymatically active proteins. However, not all category I mutant proteins were PI-G tailed to the same extent. Pre-pro-PLAP with other substituents at position 484 (threonine, proline, methionine, valine, leucine, tyrosine, tryptophan, lysine, glutamic acid, and glutamine; category II) were expressed, as well as the category I amino acids, but there was little or no processing to the PI-G-tailed form, and this latter group exhibited very low enzyme activity. The bulk of the PLAP protein produced by category II mutants and some produced by category I mutants were sequestered within the cell, apparently in the endoplasmic reticulum (ER). Most likely, certain amino acids at residue 484 are preferred because they yield better substrates for the putative "transamidating" enzyme. In transfected COS cells, at least, posttranslational PI-G-tail processing does not go to completion even for preferred substrates. Apparently PI-G tailing is a requisite for transport from the ER and for PLAP enzyme activity. Proteins that are not transamidated are apparently retained in the ER in an inactive conformation.

Conversion of placental alkaline phosphatase from a phosphatidylinositol-glycan-anchored protein to an integral transmembrane protein.

Placental alkaline phosphatase (PLAP) is normally anchored to the plasma membrane of cells by a phosphatidylinositol-glycan anchor after removal of a carboxyl-terminal peptide from the nascent enzyme. To investigate the signals required for this processing we constructed a chimeric cDNA. The latter was designed to code for a truncated precursor form of PLAP, containing the phosphatidylinositol-glycan attachment site but incapable of any form of membrane attachment, fused to a carboxyl-terminal peptide of vesicular stomatis virus glycoprotein. Expression of the PLAP-vesicular stomatis virus glycoprotein chimeric cDNA in transfected COS cells produced an enzymatically active protein that was attached to the plasma membrane, with the PLAP domain on the outer surface. Assays for the presence of phosphatidylinositol-glycan attachment proved negative, whereas an antibody assay confirmed the presence of the vesicular stomatis virus glycoprotein carboxyl-terminal peptide, leading to the conclusion that the truncated PLAP is attached to the cells by the membrane-spanning domain of the vesicular stomatis virus glycoprotein. In light of previous findings on carboxyl-terminal requirements of PLAP these studies suggest that an essential signal for correct sorting between transmembrane insertion and phosphatidylinositol-glycan attachment resides in the cytoplasmic domain.

Site-directed antibodies for probing the structure and biogenesis of phosphatidylinositol glycan-linked membrane proteins: application to placental alkaline phosphatase.

An immunological approach to the study of the structure and biogenesis of the phosphatidylinositol glycan (PI-G) membrane anchor at the carboxyl terminus of human placental alkaline phosphatase (PLAP) is described. Based on the protein sequence predicted from full length PLAP cDNA, two epitopes were chosen in the region of the carboxyl terminus for the production of site-directed antibodies. The exo site represents the last nine residues of preproPLAP, (res. 505-513), which is part of the sequence that is expected to be cleaved from the nascent protein during processing and addition of the PI-G tail. A second site, the endo sequence, was selected close to the expected carboxyl terminus in mature PI-G-tailed PLAP (res. 474-484 of proPLAP). The two peptides were synthesized, polyclonal antibodies to the conjugated peptides were prepared, and the antisera were characterized. Analytical methods for both synthetic peptides and proteins are presented. Preliminary applications to the isolation and characterization of the PI-G-linked carboxyl terminus of mature PLAP and to the characterization of nascent PLAP are described. The application of both carboxyl terminal-directed antibodies, and a third antibody directed to the amino terminus of mature PLAP, in studies employing mutant forms of PLAP and to the PI-G tailing process itself are discussed. The immunological approach used here for PLAP should be applicable generally to the study of other PI-G-tailed proteins.

Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme.

A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purified by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the alpha carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the post-translational condensation with the phosphatidylinositol-glycan.

Purification and sequence of a novel ovine adrenal medullary peptide and its precursor.

A 24 amino acid polypeptide that does not originate from (pre)proenkephalin has been isolated from ovine adrenal chromaffin granules. Its sequence is: Arg-Leu-Pro-Gly-Glu-Leu-Arg-Asn-Tyr-Leu-Asp-Tyr-Gly-Glu-Glu-Val-Gly-Glu- Glu-Ala -Ala-Arg-Gly-Val. This peptide is generated from a precursor molecule that has also been purified and partially sequenced. The proteolytic cleavage occurs at a triple Arg site. A search of the available protein sequence data banks shows very little homology to any known protein.

Entropic DNA.

The presence of mobile genetic elements without apparent biological function within genomes requires explanation. It is argued that consideration of DNA as an open thermodynamic system leads to the simple hypothesis that mobile genetic material increases the internal entropy thereby lowering the free energy of the DNA relative to DNA of the same size. This phenomenon enhances the survival of such sequences by simple structural stabilization. The consequences of this idea are discussed in terms of the proposed Bekenstein limit to the entropy to energy ratio of thermodynamic systems and biological entropy/information flow.

Protein conformation and reversed-phase high-performance liquid chromatography.

The structure of a series of proteins has been investigated by circular dichroism, fluorescence and visible spectroscopy as well as by differential scanning calorimetry under reversed-phase high-performance liquid chromatography elution conditions. These studies show that 1-propanol, a typical eluent, induces a reversible conformational change in proteins to an apparently ordered, helical form. This structural transition occurs in the range of propanol concentrations that produces elution of a particular protein. The possible relationship between this conformational change and protein elution is considered.

Purification and sequence of a non-opioid peptide derived from ovine proenkephalin: implications for possible species specific processing.

A non-enkephalin containing pentadeca peptide derived from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: Phe-Ala-Glu-Pro-Leu-Pro-Ser-Glu-Glu-Glu-Gly-Glu-Ser-Tyr-Ser (preproenkephalin 237-251) representing the amino portion of peptide B (preproenkephalin 237-268). The sequence is identical to bovine preproenkephalin 237-251, differing from the corresponding human sequence at positions 240 and 244. This peptide can be generated by a processing event common to other opioid peptides and is present in chromaffin granules in significant amounts. The presence of this peptide in substantial quantities suggests a possible difference in proenkephalin processing between the bovine and ovine adrenal medulla.

Adrenal medullary chromaffin granule peptides: two major ovine peptides and their precursor.

An octapeptide and decapeptide which are not derived from proenkephalin were isolated from ovine adrenal chromaffin granules. Their sequences are Asn-Leu-Asp -Pro-Lys-Leu-Asp-Leu and Val-Ala-Glu-Leu-Asp-Gln-Leu-Leu-His-Tyr. These two peptides were found to be derived from a single precursor peptide which has also been isolated and sequenced. The proteolytic cleavage occurs at a Lys-Arg site typical of prohormone to hormone cleavages.

A simple experimental model for hydrophobic interactions in proteins.

The compound N-cyclohexyl-2-pyrrolidone contains a substantial apolar region as well as a peptide bond-like moiety. This solvent, therefore, provides a useful model for protein interiors. Under certain conditions of temperature and salt concentration, cyclohexylpyrrolidone forms a two-phase system with water. This permits partition coefficients and subsequent free energies of transfer of amino acid side chains from cyclohexylpyrrolidone to water to be simply determined. Free energies of transfer measured in this manner for 21 amino acids are found to be substantially less than those obtained from the commonly used ethanol/water solubility model. This suggests less of a contribution of hydrophobic interactions to the stabilization of protein structure than is conventionally assumed.

Purification and sequence of an opioid peptide derived from ovine proenkephalin.

An enkephalin-containing peptide originating from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: GLY-GLY-GLU-VAL-LEU-GLY-LYS-ARG-TYR-GLY-GLY-PHE-MET (preproenkephalin 128-140) which represents a portion of peptide F (preproenkephalin 107-140). This peptide has a sequence identical to that of bovine preproenkephalin 128-140 while it differs from the corresponding human sequence in positions 129, 131 and 133.