A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor.

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI. IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.

Telehealth and telenursing using simulation for pre-licensure USA students.

An innovative simulation was used to teach pre-licensure USA nursing students about telenursing for screening, assessment, and patient education during a home visit. The students used telepresence technology to deliver nursing care to a homebound geriatric patient. After the simulation, students (N = 73) felt increased confidence with the skills needed to deliver telenursing care and in using telepresence technology. Students reported that they modified and improved their communication in order to adapt to perceived barriers posed by the new technology. As telenursing becomes more prevalent for managing patient care at a distance, nursing programs will need to incorporate educational strategies to reflect this change.

Polyvictimization and its relationship to symptoms of psychopathology in a southern European sample of adolescent outpatients.

This study examined the relationship between accumulated experiences of victimization and symptoms of psychopathology in 132 adolescent outpatients aged 12-17 years (M=14.27; SD=1.42). The Juvenile Victimization Questionnaire and the Youth Self-Report were used to analyze polyvictimization and symptoms of psychopathology, respectively. The interviews were conducted between December 2009 and May 2012. Cluster analysis identified a subgroup of polyvictimized patients (n=17) whose general psychological impairment was significantly worse and who presented significantly more externalizing and internalizing symptoms in comparison to the rest of the sample. This difference remained significant when taking into account the clinical severity of these symptoms. These results should be taken into account when assessing and treating adolescent outpatients, for whom an adequate prognosis must be made in line with their experiences and distress. Both the self-report technique and the statistical procedure used have been shown to be suitable for identifying victimization experiences in outpatients, although this new evidence requires confirmation in future research.

Migration, ethnicity and environment: HIV risk factors for women on the sugar cane plantations of the Dominican Republic.

OBJECTIVE: To determine risk factors for HIV infection among women living in the sugar cane plantation communities (bateyes) of a large private sugar cane company in the Dominican Republic. DESIGN: Cross-sectional study of sexually active female volunteers living in the bateyes. METHODS: Of 98 bateyes, 23 were randomly selected and visited by a mobile medical unit, to interview, examine and test volunteers for seroreactivity to HIV and syphilis. RESULTS: The 490 subjects ranged in age from 16 to 72 years (median, 37 years); 53% were born in Haiti, 36% in Dominican Republic bateyes, and 12% elsewhere in the Dominican Republic; 58% had no formal education; and 87% had no income. HIV seropositivity was found in 28 women (5.7%), including 8.8% of those aged < 35 years. By logistic regression analysis, HIV infection was independently associated with age < 35 years [odds ratio (OR), 4.5; P < 0.01), being single with children (OR, 4.3; P < 0.01), more than one lifetime sex partners (OR, 3.4; P = 0.06), engaging in sex during menses (OR, 3.2; P = 0.02), and self-description as a prostitute (OR, 4.4; P = 0.05)1. For Haitian women, those coming to the Dominican Republic alone were more likely to have HIV infection than those coming with a male partner. Less than 4% of women reported condom use at last intercourse. CONCLUSIONS: Women in the bateyes have a much higher rate of HIV infection than that estimated for women in the general population of Dominican Republic and a rate comparable to that of female sex workers in the Dominican Republic. AIDS prevention in the bateyes should address condom education and distribution as well as employment opportunities and education for women.

Protecting paradise: tourism and AIDS in the Dominican Republic.

This study summarizes results from six data collection instruments administered to tourists, hotel workers, and commercial sex workers (CSWs) in the Dominican Republic (D.R.). The objective of this study was to assess: 1. how HIV/AIDS may affect tourism; 2. how tourists are likely to react to prevention campaigns; and 3. how tourism may affect the spread of HIV/AIDS. It was found that an overwhelming proportion of tourists did not consider the prevalence of HIV to be a factor when making their travel plans, and that most did not consider themselves at greater risk of becoming infected while on holiday than when they were at home. This study determined that the spread of HIV/AIDS was unlikely to affect the demand for tourism services in the D.R. The study also found that most tourists would respond positively to an HIV/AIDS prevention campaign and would not be discouraged from visiting the D.R. because of such campaigns. Those most receptive to prevention efforts were also those who felt they were at highest risk, according to study data. Finally, it was determined that while most tourists probably do not engage in high risk activities, there were some male and female tourists who do engage in sexual encounters with multiple Dominican CSWs and hotel employees. These encounters represent a risk to the health and economic development of the D.R., as well as to tourists and their other sexual partners. Based on these findings, it is recommended that in order to minimize the potential social and economic impact of HIV/AIDS in the D.R., prevention messages need to reach a number of groups which have not yet been adequately targeted. These groups include tourists, with a special emphasis on 'sex tourists', and hotel employees, with a special emphasis on entertainment staff.

Sexual behaviors and risk factors for HIV infection among men who have sex with men in the Dominican Republic.

OBJECTIVE: To describe self-reported types of sexual identity of men who have sex with men (MSM) in the Dominican Republic, assess sociodemographics and behavioral characteristics, and measure the prevalence of HIV-1 and syphilis. DESIGN: Cross-sectional study of MSM recruited from a variety of community settings. METHODS: A total of 354 men agreed to participate after giving verbal informed consent. Information was obtained using a standardized questionnaire assessing demographics and AIDS-relevant information. Blood was obtained for HIV and syphilis testing. RESULTS: Five main sexual identity groups emerged: cross dressers, homosexuals, gigolos, bisexuals and heterosexuals. Receptive anal and oral intercourse were commonly reported by men self-identifying as cross dressers or homosexuals, whereas nearly all of the remaining three groups practiced only insertive intercourse. Sexual contact with women was also commonly reported; overall, consistent condom use was infrequent. HIV antibodies were detected in 11.0% and serologic evidence of syphilis was found in 7.3%. Factors independently associated with HIV infection included serologic evidence of syphilis, having visited at least one of four local brothels in 1975-1985, and having had receptive anal intercourse with four or more partners in the last 12 months. CONCLUSIONS: Syphilis, sexual practices and social context of sex (commercial sex), rather than sexual identity per se, were associated with HIV infection. The complex social networks of MSM in this setting, the tendency to practice either insertive or receptive sex, but not both, infrequent condom use, high rates of syphilis and the frequency of sex with women need to be taken into account for targeted HIV prevention programs to be successful.

Interaction of sphingomyelinase with sphingomyelin analogs modified at the C-1 and C-3 positions of the sphingosine backbone.

In this study, analogs differing at the C-1 or C-3 position of the sphingosine backbone of sphingomyelin were examined in neutral pH-optimum sphingomyelinase assays. Two analogs modified at the C-1 position, ceramide-1-phosphate and ceramide-1-phosphoethanol-N,N-dimethylamine, could act as modest substrates but showed no ability to inhibit the reaction when egg sphingomyelin was used as the substrate. Four analogs of sphingomyelin differing at the C-3 position were used in which the hydroxyl group was replaced by a hydrogen atom (to give a deoxy-sphingomyelin analog), or with a O-methyl, O-ethyl or O-tetrahydropyranyl group. The deoxy analog showed no ability to compete with substrate of sphingomyelinase nor could it be hydrolyzed by the enzyme, suggesting that the hydroxyl group is a required substituent for the substrate. The 3-O-methyl and 3-O-ethyl-sphingomyelin analogs were inhibitors, with IC50 values of 50 microM and 140 microM, respectively at standard assay conditions. However, when the rat brain acidic pH-optimum sphingomyelinase was used, no inhibition by the 3-O-methyl analog could be detected. The size of the alkyl group on the ether moiety was important, as shown by the inability of 3-O-tetrahydropyranyl-sphingomyelin to compete with substrate of neutral pH-optimum sphingomyelinase.

Characterization of the neutral pH-optimum sphingomyelinase from rat brain: inhibition by copper II and ganglioside GM3.

A neutral pH-optimum sphingomyelinase (N-SMase), solubilized from rat brain membranes, was characterized with respect to metal and membrane lipid effects. Chromatofocusing chromatography, which separates proteins according to pI, showed two N-SMase activities. One eluted at pH 4.7 and the other required 0.4 M NaCl before elution. Kinetically, the two preparations appeared similar. The N-SMase eluting at pH 4.7 was most extensively studied here. Of the phospholipids studied, only phosphatidylserine showed any influence on N-SMase and that was to increase its activity by as much as 50%. Neither serine nor phosphatidic acid had any effect. Of the cations tested, none was able to replace Mg2+ as a required activator. However, it was found that several metals were inhibitory, with Cu2+ being most effective (IC50 = 5 microM). Gangliosides, particularly the monosialoganglioside, GM3 (IC50 approximately 50 microM), inhibited N-SMase. Other glycolipids showed little effect on activity, even the immediate precursor to GM3 - lactosylceramide. The ganglioside sugar, N-acetylneuraminic acid, also had no effect on N-SMase activity. None of these inhibitors affected the acidic pH-optimum sphingomyelinase. Other sphingolipid compounds such as ceramide - the enzymatic product - and sphingosylphosphorylcholine (lysosphingomyelin) showed no capacity to inhibit N-Smase, implying that the enzyme may have a selective substrate-binding site.

Interaction of cholesterol with conformationally restricted phospholipids in vesicles.

The interaction of cholesterol with conformationally restricted analogs of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in the liquid-crystalline phase has been studied in vesicles. These analogs contain one of three cyclopentane triols in place of the glycerol moiety found in natural phospholipids and make possible an analysis of whether a limitation of the conformational mobility in the glycerol backbone region affects the interaction with cholesterol. When cholesterol was incorporated into vesicles from cyclopentanoid phospholipids in which the acyl group vicinal to the head group is trans, the first-order rate constant for Cl- efflux is decreased similarly to that in vesicles from 'natural' DPPC or DPPG (about 50%). However, when the head group is in the unnatural 2 position, cholesterol has a much smaller effect on the rate of Cl- efflux (a decrease of about 20%). Cholesterol decreased the rate constants for valinomycin-mediated 86Rb+ efflux from vesicles of the cyclopentanoid PC analogs and of DPPC to a similar extent. The half-time values for spontaneous intervesicle cholesterol exchange were not markedly different using vesicles prepared with the natural glycerophospholipids and with the cyclopentano-phospholipids, suggesting that the geometrical orientation of the acyl chains or the head group has little influence on cholesterol desorption from the lipid/water interface.

An evaluation of the user acceptability of chlorhexidine handwash formulations.

Four similar-looking preparations containing 4% chlorhexidine ('Hibiscrub', Surgiscrub', 'Uniscrub', 'Macrocide') were compared in a single-blind, randomized study in a group of 73 volunteers. The volunteers washed their hands with each preparation ten times over 9 h, one preparation day-1 on each of four days over two weeks. The acceptability of the agents was assessed using visual analogue scales which were analysed by paired t-test. There were marked differences between the products. 'Uniscrub' smelled worse (P less than 0.003) and felt worse than the others (P less than 0.003). 'Hibiscrub' lathered well but 'Macrocide' was found easiest to rinse off. 'Hibiscrub' and 'Surgiscrub' were the least irritant and had the best visual appearance. Despite apparent similarities, these chlorhexidine formulations varied in acceptability to users.

Binding and inhibition studies on lipocortins using phosphatidylcholine vesicles and phospholipase A2 from snake venom, pancreas, and a macrophage-like cell line.

Studies are reported on the inhibition of phospholipase A2 (PLA2) from porcine pancreas, cobra (Naja naja) venom, and the P388D1 macrophage-like cell line by human recombinant lipocortin I and bovine lung calpactin I. Membrane vesicles prepared from 1-stearoyl,2-arachidonoyl phosphatidylcholine (PC) and other PCs were utilized as substrate. Binding studies using sucrose flotation gradients showed that both lipocortin I and calpactin I bind to these vesicles although less tightly than to vesicles prepared from anionic phospholipids or fatty acids. Binding to PC was somewhat enhanced by Ca2+. Inhibition of cobra venom PLA2 was not observed when PC vesicles were used as substrate but was when dipalmitoyl phosphatidylethanolamine was used. Both the pancreatic and macrophage enzymes were inhibited when acting on PC. Interestingly, the inhibition of the macrophage enzyme toward PC depended on the fatty acid attached to the sn-2 position of PC with arachidonate greater than oleate greater than palmitate. Inhibition was also highest at low [PC]; these inhibition results can be explained by the "substrate depletion model" (Davidson, F. F., Dennis, E. A., Powell, M., and Glenney, J. (1987) J. Biol. Chem. 262, 1698-1705). Experimental and theoretical considerations suggest that the in vitro inhibition by lipocortins of this macrophage PLA2 from a cell that makes lipocortin and is active in prostaglandin production is due to effects on substrate availability rather than direct inhibition.

Inhibition studies on the membrane-associated phospholipase A2 in vitro and prostaglandin E2 production in vivo of the macrophage-like P388D1 cell. Effects of manoalide, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacyl bromide.

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostaglandin production in the intact cell from which it is derived.

Cyclopentanoid analogs of phosphatidylcholine: susceptibility to phospholipase A2.

Six isomers of dipalmitoylcyclopentanetriol phosphocholine (cyclopentano-lecithin) were tested as potential substrates for phospholipase A2. Since each of these analogs possesses a configuration that mimics a narrow range of conformations of a glycerophospholipid molecule, the analogs were used to assess the enzyme's conformational requirements. Studies showed that all of the analogs containing the phosphocholine at the C-1 (or C-3) position could be hydrolyzed, while only one of the three analogs that contains the polar head group at the C-2 position was susceptible. Kinetic studies, however, revealed that only the all-trans-(1,3/2-1P)-cyclopentano-lecithin gave initial rates of hydrolysis that were measurable by pH-stat. Acyl group specificity of the enzyme towards the all-trans isomer was determined with an analog was acyl groups were distinguishable. The synthesis of this mixed-acid-cyclopentano-PC is described herein. When this analog was enzymatically assayed, results unequivocally showed the enzyme to be specific for C-2 acyl hydrolysis. This specificity, and data showing that the all-trans analog is stereospecifically hydrolyzed, indicate that it is acted on in an analogous manner to dipalmitoylphosphatidylcholine. These studies indicate that although the configuration of the analog is not necessarily a prerequisite for hydrolysis, there does appear to be an optimal spatial orientation for enzymatic activity. The analogy between the susceptibilities of all-trans-(1,3/2-1P)-cyclopentano-lecithin and glycero-lecithin suggests that the conformation of the glycero-lecithin during phospholipase A2-mediated hydrolysis may be best simulated by the all-trans orientation of C-O bonds in the artificial substrate.

Probing the role of substrate conformation in phospholipase A2 action on aggregated phospholipids using constrained phosphatidylcholine analogues.

Phospholipase A2s hydrolyze aggregated phospholipid substrates much more rapidly than dispersed monomeric ones. Whether this is a consequence of interface-associated conformational changes of the enzyme or of the substrate, or of both, remains a key question in lipid enzymology. This problem is addressed herein using a rationally designed probe of substrate conformation. (1,3/2)-1-O-(phosphorylcholine)-2,3-O-dihexanoylcyclopentane-1,2,3 -triol is a novel short chain phosphatidylcholine analogue in which the glycerol-like backbone is part of a five-membered ring and therefore covalently constrained within a small defined range of conformations. To the extent that the constrained analogue resists aggregation-associated conformational changes, it provides a means for assessing the contribution of such changes to phospholipase A2 action on aggregated phospholipids. The monomeric (-)-cyclopentanoid analogue is a substrate for phospholipase A2s from Naja naja naja venom. However, when this constrained phospholipid is aggregated, its hydrolysis rate is not enhanced, in contrast to its unconstrained counterpart, 1,2-dihexanoyl-sn-glycero-3- phosphorylcholine. This lack of activation was not caused by a failure of the enzyme to bind the micellar, constrained analogue. While the constrained analogue does not show interfacial activation, it does show the activation of phosphatidylethanolamine hydrolysis typical of phosphorylcholine-containing lipids. Hence, these results strongly support the contention that specific packing-induced conformations of aggregated substrate play a substantial role in the large interfacial activations observed with phospholipase A2.

Kinetic analysis of the Ca2+-dependent, membrane-bound, macrophage phospholipase A2 and the effects of arachidonic acid.

The kinetics of the Ca2+-dependent, alkaline pH optimum, membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line were studied using various phosphatidylcholine (PC) and phosphatidylethanolamine (PE) substrates. This enzyme exhibits "surface dilution kinetics" toward PC in Triton X-100 mixed micelles, and the "dual phospholipid model" was found to adequately describe its kinetic behavior. With substrate in the form of sonicated vesicles, the dual phospholipid model should give rise to Michaelis-Menten type kinetics. However, the hydrolysis of dipalmitoyl-PC, 1-palmitoyl-2-oleoyl-PC, and 1-stearoyl-2-arachidonoyl-PC vesicles exhibited two distinct activities. Below 10 microM, the data appeared to follow Michaelis-Menten behavior, while at higher concentrations, the data could best be fit to a Hill equation with a Hill coefficient of 2. These PCs had Vmax values for the low substrate concentration range of 0.2-0.6 nmol min-1 mg-1 and Km values of 1-2 microM. At the high substrate concentration range, the Vmax values were between 5 and 7 nmol min-1 mg-1. PC containing unsaturated fatty acids had an apparent Km, determined from the Hill equation, of about 15 microM, while the apparent Km of dipalmitoyl-PC was 0.6 microM. When 70% glycerol was included in the assays, a single Michaelis-Menten curve was obtained for both dipalmitoyl-PC and 1-stearoyl,2-arachidonoyl-PC. Possible explanations for these kinetic results include reconstitution of the membrane-bound phospholipase A2 in the phospholipid vesicle or the enzyme has tow distinct phospholipid binding function. The kinetics for both dipalmitoyl-PC and dipalmitoyl-PE hydrolysis in vesicles was very similar, indicating that the enzyme does not greatly prefer one of these head groups over the other. The enzyme also showed no preference for arachidonoyl containing phospholipid. Enzymatic activity toward PC containing saturated fatty acids was linear to about 15% hydrolysis while the hydrolysis of PC containing unsaturated fatty acids was linear to only about 5%. This loss of linearity was due to inhibition by released unsaturated fatty acids. Arachidonic acid was found to be a competitive inhibitor of dipalmitoyl PC hydrolysis with a K1 of 5 microM. This tight binding suggests a possible in vivo regulatory role for arachidonic acid. Three compounds of the arachidonic acid cascade, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, showed no inhibition of enzymatic activity.

Synthesis and some properties of constrained short-chain phosphatidylcholine analogues: (+)- and (-)-(1,3/2)-1-O-(phosphocholine)2,3-O- dihexanoylcyclopentane-1,2,3-triol.

Reported herein is the synthesis of (+)- and (-)-(1,3/2)-1-O-(phosphocholine)-2,3-O-dihexanoylcyclopentane-1,2, 3-triol. These are the enantiomers of a contrained analogue of dihexanoylphosphatidylcholine in which the glycerol backbone is replaced by all-trans cyclopentane-1,2,3-triol. Evidence is presented to demonstrate that the (-)-enantiomer is a substrate for phospholipase A2 (PLA2) (Crotalus atrox) while the (+)-enantiomer is not. This strict enantiomeric (and positional) specificity was exploited in conjunction with a novel application of DEAE-cellulose column chromatography, to achieve racemic resolution with an excellent yield. The constrained backbone geometry, and the experimentally accessible critical micellar concentration (CMC) of these analogues should render them useful probes for assessing the contribution of substrate conformation and flexibility to the catalytic efficiency of PLA2.

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.