Sheep Seminal Vesicles Research Articles

Determination of the structure of the carbohydrate chains of prostaglandin endoperoxide synthase from sheep.

Prostaglandin endoperoxide synthase was isolated from sheep seminal vesicles. Sugar analysis of the glycoprotein revealed the presence of mannose and N-acetylglucosamine only. The carbohydrate moiety was released from the polypeptide backbone by hydrazinolysis. After re-N-acetylation and reduction, the resulting mixture of oligosaccharide-alditols was fractionated on Bio-Gel P-4 and their structures were investigated by 500-MHz1H-NMR spectroscopy. The carbohydrate chains turned out to be of the oligomannoside type containing six to nine mannose residues. The largest and most abundant compound was established to be: (formula; see text) For the smaller structures heterogeneity occurs with respect to the outer alpha(1----2)-linked mannose residues. Furthermore, a small amount of Man6GlcNAc-ol (artefact of the hydrazinolysis procedure) was detected by 1H-NMR spectroscopy and fast atom bombardment mass spectrometry.

Phospholipid actions on PGHS-1 and -2 cyclooxygenase kinetics

Cyclooxygenase (COX) catalysis by prostaglandin H synthase (PGHS) is a key control step for regulation of prostanoid biosynthesis. Both PGHS isoforms are integral membrane proteins and their substrate fatty acids readily partition into membranes, but the impact of phospholipids and lipid membranes on COX catalysis and the actions of COX inhibitors are not well understood. We have characterized the COX kinetics and ibuprofen inhibition of the purified PGHS isoforms in the presence of phosphatidylcholine (PC) with varying acyl chain structure and physical state. PC was found to directly inhibit COX activity, with non-competitive inhibition by PC monomers binding away from the COX active site and competitive inhibition by micellar/bilayer forms of PC due to sequestration of the arachidonate substrate. Competitive inhibition by native membranes was observed in a comparison of COX kinetics in sheep seminal vesicle microsomes before and after solubilization of PGHS-1. PC liposomes significantly increase the inhibitory potency of ibuprofen against both PGHS isoforms without changing the reversible character of ibuprofen action or requiring binding of PGHS to the liposomes. These results suggest a useful conceptual framework for analyzing the complex interactions among the PGHS proteins, substrates, inhibitors and phospholipid.

In vitro effects of E3040, a dual inhibitor of 5-lipoxygenase and thromboxane A 2 synthetase, on eicosanoid production

In vitro pharmacological profiles of E3040, 6-hydroxy-5, 7-dimethyl-2-(methylamino)-4-(3-pyridylmethyl) benzothiazole were investigated. Against the 5-lipoxygenase activity of rat basophilic leukemia cells, E3040 and zileuton (a 5-lipoxygenase inhibitor) had an IC 50 of 0.23 and 0.93 μM, respectively. Against the thromboxane A 2 synthetase activity of human platelets, E3040 had an IC 50 of 0.01 μM, which was comparable to that of OKY-1581 (sodium ( E)-3-[4-(3-pyridylmethyl) phenyl]-2-methylacrylate, a thromboxane A 2 synthetase inhibitor). Against cyclooxygenase activity of sheep seminal vesicles, E3040 showed no inhibition (IC 50, >300 μM). Sulfasalazine and 5-aminosalicylic acid, therapeutic drugs for inflammatory bowel disease, inhibited 5-lipoxygenase activity with an IC 50 of 293 and 970 μM, respectively. Sulfasalazine inhibited thromboxane A 2 synthetase activity with an IC 50 of 20 μM. In rat peritoneal leukocytes, E3040 inhibited leukotriene B 4 and thromboxane B 2 production with an IC 50 of 0.17 and 0.24 μM, respectively. E3040 inhibited leukotriene B 4 production in human neutrophils and thromboxane B 2 production in human platelets (IC 50 of 0.21 and 0.09 μM, respectively). These results indicated that E3040 potently inhibited 5-lipoxygenase and thromboxane A 2 synthetase and blocked leukotriene B 4 and thromboxane B 2 production in rat peritoneal and human blood cells.

Existence of endogenous inhibitor(s) of prostaglandin endoperoxide H synthase activities in murine NIH3T3 fibroblasts

The production of prostaglandins (PGs) is regulated by several processes, i.e. arachidonic acid release, prostaglandin endoperoxide H synthase (PGHS) activity and its induction. In the present study, we investigated the possibility that inhibitor(s) of PGHS activities exist endogenously and regulate PG production. NIH3T3 cell, a cloned murine fibroblast, expressed PGHS-1 under unstimulated conditions and induced PGHS-2 protein under stimulation by serum. When PGHS activity was measured by individual intact cell assay, there was heterogeneity in PGHS activities in individual cells after serum treatment. We isolated and cultured many cloned cells which showed different PGHS activities after serum treatment. In these cells, however, there was no difference in the expression of PGHS-1 and PGHS-2 mRNA. The protein level of PGHS-1 and PGHS-2 was also not different in these cloned cells. The lysate obtained from the cells that showed lower PGHS activity after the serum treatment suppressed PGHS-1 activities of sheep seminal vesicle and PGHS-2 activity in purified ovine placenta. The suppression by this lysate was stronger than that by the lysate obtained from the cells which showed higher PGHS activity after the serum treatment. The production of the inhibitor(s) was up-regulated by the serum treatment and abolished by actinomycin D and cycloheximide during the serum treatment. From these results, we suggest that there is some endogenous inhibitor(s) of PGHS-1 and -2 activities in NIH3T3 cells, and that the inhibitor(s) is induced by serum.

Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target.

Human prostaglandin (PG) E synthase (EC 5.3.99.3) is a member of a recently recognized protein superfamily consisting of membrane associated proteins involved in eicosanoid and glutathione metabolism (the MAPEG family). Previous designations of the protein are PIG12 and MGST1-L1. PGE synthase was expressed in Escherichia coli, and both cytosolic and membrane fractions were prepared. Western blot analysis specifically detected a 15- to 16-kDa protein in the membrane fraction. Both fractions were incubated with prostaglandin H2 in the presence or absence of reduced glutathione. The membrane but not the cytosolic fraction was found to possess high glutathione-dependent PGE synthase activity (0.25 micromol/min/mg). The human tissue distribution was analyzed by Northern blot analysis. High expression of PGE synthase mRNA was detected in A549 and HeLa cancer cell lines. Intermediate level of expression was demonstrated in placenta, prostate, testis, mammary gland, and bladder whereas low mRNA expression was observed in several other tissues. A549 cells have been used as a model system to study cyclooxygenase-2 induction by IL-1beta. If A549 cells were grown in the presence of IL-1beta, a significant induction of the PGE synthase was observed by Western blot analysis. Also, Western blot analysis specifically detected a 16-kDa protein in sheep seminal vesicles. In summary, we have identified a human membrane bound PGE synthase. The enzyme activity is glutathione-dependent, and the protein expression is induced by the proinflammatory cytokine IL-1beta. PGE synthase is a potential novel target for drug development.

Molecular model of the interaction between nimesulide and human cyclooxygenase-2.

The cyclooxygenase-2 (COX-2) isoenzyme is a key target for COX-2-selective non-steroidal anti-inflammatory drugs (NSAIDs). An important difference in binding of nimesulide compared with non-selective NSAIDs appears to involve the amino acid at position 523 of the enzyme. Replacement of valine with isoleucine at this position provides access to a binding site that is larger in COX-2 than in COX-1. Nimesulide appears to exploit this enlarged binding site for establishing a number of favourable contacts with the enzyme that lead to selective inhibition of COX-2. We made these conclusions from a three-dimensional molecular model of the active site of human COX-2, constructed using the X-ray coordinates of COX-1 from sheep seminal vesicles and COX-2 from mouse fibroblasts as templates, with the aid of sequence alignment methods and molecular modelling techniques. The resulting model was refined, and the active site was probed for regions of steric and electrostatic complementarity for ligand binding. Docking studies were then undertaken with many different nimesulide conformers, a family of which could establish very favourable interactions with the NSAID binding site of human COX-2 by exploiting the extra space made available by the isoleucine/valine replacement. The stability of the resulting complexes was studied by simulating molecular dynamics.

ER-34122, a novel dual 5-lipoxygenase/cyclooxygenase inhibitor with potent anti-inflammatory activity in an arachidonic acid-induced ear inflammation model.

To investigate the effect of ER-34122, a novel pyrazole derivative, on 5-lipoxygenase (LOX) and cyclooxygenase (COX) metabolite production in vitro, ex vivo and in vivo. In vitro, lysate of rat basophilic leukemia cells, the microsome fraction of sheep seminal vesicles, human polymorphonuclear leukocytes, human synovial cells, and human monocytes. Ex vivo and in vivo, male Balb/c mice or SD rats. In ex vivo study, ER-34122 (0.03-1 mg/kg) was orally administered 1 h before withdrawal of blood samples. In carrageenin-induced paw edema, ER-34122 (3-100 mg/kg) and indomethacin (1-10mg/kg) were orally administered 1 h before carrageenin injection. In arachidonic acid-induced ear inflammation, ER-34122 (0.3-10mg/kg), zileuton (10-100mg/kg) and indomethacin (0.3-3mg/kg) were orally administered 1 h before arachidonic acid application. 5-Hydroxyeicosatetraenoic acid and other eicosanoids were determined by using an HPLC method and enzyme immunoassay, respectively. Rat hind paw edema and mouse ear edema were assessed by measuring paw volume and ear thickness, respectively. Myeloperoxidase (MPO) activity and eicosanoid content of the ear tissue were also determined. ER-34122 inhibited both LOX and COX product generation in vitro, and ex vivo. ER-34122 and indomethacin inhibited carrageenin-induced paw edema formation. In the arachidonic acid-induced ear inflammation, ER-34122 inhibited inflammatory responses (edema formation and MPO accumulation) as well as eicosanoids (LTB4, LTC4 and PGE2) generation. A representative LOX inhibitor, zileuton, also inhibited these inflammatory responses, while a COX inhibitor, indomethacin, did not suppress them though it completely inhibited PGE2 generation. The anti-inflammatory characteristics of ER-34122 are considered to be superior to those of COX inhibitors such as indomethacin, because in addition to its inhibitory activity on the COX pathway, ER-34122 inhibits LOX products generation, as revealed by the inhibition of edema formation or polymorphonuclear leukocyte infiltration in the arachidonic acid-induced ear inflammation model.

Alpha-viniferin: a prostaglandin H2 synthase inhibitor from root of Carex humilis.

An inhibitor on cyclooxygenase activity of prostaglandin H2 synthase was purified from the root of Carex humilis Leyss (Cyperaceae) by a variety of column chromatographic methods. As a result of the structure analysis by FAB-mass, 1H-NMR, and 13C-NMR spectral data, the active compound was identified as (+)-alpha-viniferin, an oligomeric stilbene characterized originally from Caragana chamlagu Lamarck (Leguminosae). (+)-alpha-Viniferin exhibited a dose-dependent inhibition on cyclooxygenase activity, where 50% of inhibition (IC50) was shown at a final concentration of about 7 microM. Resveratrol, a putative building block of oligomeric stilbenes, also inhibited the cyclooxygenase activity. The inhibitory potency of (+)-alpha-viniferin was about 3- to 4-fold stronger than that of resveratrol on cyclooxygenase activity of prostaglandin H2 synthase partially purified from sheep seminal vesicles.

Inhibitory effects of hydroxystilbenes on cyclooxygenase from sheep seminal vesicles.

Inhibitory effects of hydroxystilbenes on cyclooxygenase from sheep seminal vesicles.

Effect of ebselen on IL-1-induced alterations in cartilage metabolism.

To evaluate the effect of the antioxidant-like anti-inflammatory agent, ebselen, on cartilage proteoglycan degradation and to determine whether its cartilage protectant activity is related to its antioxidant activity. Cartilage in organ culture was stimulated with interleukin-1 (IL-1), and proteoglycan degradation was assessed by measuring the amount of sulfated glycosaminoglycan released into the media, proteoglycan synthesis evaluated by [35S]-sulfate incorporation, and prostaglandin E2 (PGE2) release determined by radioimmunoassay (RIA). Glutathione peroxidase (GSH-Px) activity was evaluated in a coupled test system using NADPH/GSSG reductase as an indicator and cyclooxygenase activity was evaluated using sheep seminal vesicle prostaglandin synthase. Ebselen caused a concentration-dependent inhibition of IL-1-stimulated proteoglycan degradation with an IC50 of 4.7 microM. Cartilage PGE2 release was also reduced in the presence of ebselen (IC50 = 6.2 microM). However, at concentrations up to 100 microM, ebselen had no effect on the inhibition of proteoglycan synthesis by IL-1. Induction of proteoglycan breakdown was also inhibited by a sulfur analog of ebselen. This analog was devoid of GSH-Px activity and was 50-fold less potent in cyclooxygenase inhibitory activity, but was equipotent to ebselen in inhibiting cartilage degradation. Ebselen, unlike other NSAIDs, blocks cartilage proteoglycan breakdown without inhibiting proteoglycan synthesis. This effect is independent of its GSH-Px activity and its ability to inhibit cyclooxygenase and PGE2 production. Therefore, this compound may provide a new mechanism for protecting cartilage matrix from degradative factors in arthritic joints.

Biologically active alkamide from the lichen Stereocaulon alpinum

The presence of an alkamide in lichens is reported for the first time. The alkamide was isolated from Stereocaulon alpinum Laur. and identified through spectroscopic analysis as 9-cis-octa-decenamide (1). With reference to previously known activity of alkamides from both the plant and animal kingdoms on inflammatory processes, 1 was tested for in vitro inhibitory activity against the key enzymes of the major pathways involved in arachidonate metabolism. The compound was shown to exhibit moderate dose-dependent inhibitory activity against cyclooxygenase from sheep seminal vesicle microsomes (IC(50) = 64.3 μM) whilst showing only slight activity against 5-lipoxygenase from porcine leucocytes.

Pharmacological profile of JTE-522, a novel prostaglandin H synthase-2 inhibitor, in rats.

The antinociceptive, antipyretic, and anti-inflammatory effects of JTE-522, a novel selective prostaglandin H synthase (PGHS)-2 inhibitor, were examined in rats. Sheep seminal vesicle PGHS-1 and placenta PGHS-2 were used for in vitro assay, while for in vivo experiments, male rats (4-8 weeks old) were used. JTE-522 and reference compounds (0.01-100 microM) were subjected to enzyme assay. JTE-522 (0.3-30 mg/kg) and indomethacin (0.3-10 mg/kg) were administered orally. JTE-522 inhibited PGHS-2 (IC50: 0.64 microM) without affecting PGHS-1 activity at 100 microM. In rats with yeast-induced hyperalgesia, JTE-522 showed a dose-dependent antinociceptive effect (ED50: 4.4 mg/kg). In rats with yeast-induced pyrexia, JTE-522 significantly reversed the pyrexic response (ED50: 3.9 mg/kg). Orally administered JTE-522 dose-dependently inhibited carrageenin-induced rat paw edema (ED30: 4.7 mg/kg). In rats with adjuvant-induced arthritis, JTE-522 showed a significant inhibitory effect at daily doses of 0.3-3 mg/kg. JTE-522 did not cause severe gastric lesions at oral doses up to 300 mg/kg. Our results indicate that the selective PGHS-2 inhibitor JTE-522 may represent a novel type of anti-inflammatory drug without adverse effects on the gastrointestinal tract. JTE-522 may thus be a promising agent for treating both acute inflammatory disease and chronic inflammatory diseases such as rheumatoid arthritis.

Inhibitory effects of baeomycesic acid from the lichen Thamnolia subuliformis on 5-lipoxygenase in vitro

Baeomycesic acid (1), a β-orcinol depside isolated from the lichen Thamnolia subuliformis (Ehrh.) W. Culb. was tested for in vitro inhibitory effects on arachidonate metabolism. Results showed that baeomycesic acid has potent dose-dependent inhibitory effects on 5-lipoxygenase isolated from porcine leucocytes, with an IC(50) value of 8.3 μM. Structure-activity relationships are discussed in comparison to other phenolic lichen constituents. Baeomycesic was inactive when tested for in vitro inhibitory effects against cyclooxygenase isolated from sheep seminal vesicle microsomes.

Isolation and characterization of an enzyme from sheep seminal vesicles that catalyzes the glutathione-dependent reduction of prostaglandin H2 to prostaglandin F2 alpha.

An enzyme which catalyzes the direct 2-electron reduction of prostaglandin H2 to prostaglandin F2 alpha has been purified from the microsomes of sheep seminal vesicles. This enzyme, called prostaglandin endoperoxide reductase, was found to be a monomer of 16,500 molecular mass. The activity of the enzyme was dependent on reduced glutathione, enhanced by heat-treatment, and inhibited by sulfhydryl reagents. The enzyme is not a glutathione S-transferase nor does it utilize prostaglandin D2 as a substrate, and thus is distinct from previously characterized prostaglandin F2 alpha biosynthetic enzymes. The protein also catalyzes the reduction of cumene hydroperoxide, but not hydrogen peroxide. Thus, this microsomal prostaglandin endoperoxide reductase may play an important role in the synthesis of prostaglandin F2 alpha in some tissues.

5-Lipoxygenase inhibitors: synthesis and structure-activity relationships of a series of 1-aryl-2H,4H-tetrahydro-1,2,4-triazin-3-ones.

Synthetic routes were developed to access a variety of novel 1-aryl-2H,4H-tetrahydro-1,2,4-triazin-3-one analogs which were evaluated as 5-lipoxygenase (5-LO) inhibitors. The parent structure, 1-phenylperhydro-1,2,4-triazin-3-one (4), was found to be a selective inhibitor of 5-LO in broken cell, intact cell, and human blood assays with IC50 values of 5-21 microM. In a rat anaphylaxis model, 4 blocked leukotriene formation with an ED50 = 7 mg/kg when administered orally. Compound 4 exhibited selectivity for inhibition of 5-LO with little activity against related enzymes: 12-LO from human platelets, 15-LO from soybean, and cyclooxygenase (COX) from sheep seminal vesicle. In pilot subacute toxicity testing, 4 did not produce methemoglobinemia in rats (400 mg/kg po daily for 9 days) or in dogs (200 mg/kg po daily for 28 days). These results indicated that the triazinone structure provided a 5-LO inhibitor template devoid of the toxicity problems observed in the related phenidone (1) and pyridazinone (3) classes of 5-LO inhibitors. The parent compound 4 is a selective, orally bioavailable 5-LO inhibitor which can serve as a useful reference standard for in vivo pharmacological studies involving leukotriene-mediated phenonmena.

Analytical and pharmacological studies on Mahonia aquifolium

Alcoholic extracts of the antipsoriatic Mahonia aquifolium drug which were analysed for their qualitative and quantitative alkaloid content, showed an in vitro inhibiting effect on cyclo-oxygenase (CO) of sheep seminal vesicles and 5-lipoxygenase (5-LO) of porcine leucocytes. The same extracts exhibited also immunostimulating activities in two phagocytosis assays. In contrast the major alkaloids of Mahonia aquifolium extracts berberine, palmatine, magnoflorine and jatrorrhizine were found to be inactive at all in both antiinflammatory assays, suggesting a different mechanism of action for the alkaloids or further not yet detected compounds in the extract.

Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase.

Constitutive cyclooxygenase (COX-1; prostaglandin-endoperoxide synthase, EC 1.14.99.1) is present in cells under physiological conditions, whereas COX-2 is induced by some cytokines, mitogens, and endotoxin presumably in pathological conditions, such as inflammation. Therefore, we have assessed the relative inhibitory effects of some nonsteroidal antiinflammatory drugs on the activities of COX-1 (in bovine aortic endothelial cells) and COX-2 (in endotoxin-activated J774.2 macrophages) in intact cells, broken cells, and purified enzyme preparations (COX-1 in sheep seminal vesicles; COX-2 in sheep placenta). Similar potencies of aspirin, indomethacin, and ibuprofen against the broken cell and purified enzyme preparations indicated no influence of species. Aspirin, indomethacin, and ibuprofen were more potent inhibitors of COX-1 than COX-2 in all models used. The relative potencies of aspirin and indomethacin varied only slightly between models, although the IC50 values were different. Ibuprofen was more potent as an inhibitor of COX-2 in intact cells than in either broken cells or purified enzymes. Sodium salicylate was a weak inhibitor of both COX isoforms in intact cells and was inactive against COX in either broken cells or purified enzyme preparations. Diclofenac, BW 755C, acetaminophen, and naproxen were approximately equipotent inhibitors of COX-1 and COX-2 in intact cells. BF 389, an experimental drug currently being tested in humans, was the most potent and most selective inhibitor of COX-2 in intact cells. Thus, there are clear pharmacological differences between the two enzymes. The use of such models of COX-1 and COX-2 activity will lead to the identification of selective inhibitors of COX-2 with presumably less side effects than present therapies. Some inhibitors had higher activity in intact cells than against purified enzymes, suggesting that pure enzyme preparations may not be predictive of therapeutic action.

Suppression of inflammatory responses to 12-O-tetradecanoyl-phorbol-13-acetate and carrageenin by YM-26734, a selective inhibitor of extracellular group II phospholipase A2.

1. YM-26734 [4-(3,5-didodecanoyl-2,4,6-trihydroxyphenyl)-7-hydroxy-2-(4-hydroxyph eny l) chroman] dose-dependently inhibited the activities of extracellular phospholipase A2 (PLA2): rabbit platelet-derived group II and porcine pancreas-derived group I PLA2, with IC50 values of 0.085 (0.056-0.129, n = 5) and 6.8 (5.0-9.6, n = 5) microM, respectively. 2. In contrast, YM-26734 did not reduce the activity of intracellular PLA2 prepared from mouse macrophages, which preferentially hydrolyzed arachidonoyl phospholipids at concentrations up to 50 microM. YM-26734 also showed no effect against either sheep seminal vesicle cyclo-oxygenase or rat leukocyte 5-lipoxygenase. 3. Linewater-Burk analysis showed that YM-26567-1 behaved as a competitive inhibitor of group II PLA2 derived from rabbit platelets, with a Ki value of 48 nM. 4. In mice, YM-26734 inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microgram/ear)-induced ear oedema in a dose-dependent manner, with ED50 values of 45 (30-67) micrograms/ear (n = 5) and 11 (4-32) mg kg-1, i.v. (n = 5), but did not decrease arachidonic acid (4 mg/ear)-induced ear oedema at 1 mg/ear and 30 mg kg-1, i.v. 5. In rats, the accumulation of exudate fluids and leukocytes in the pleural cavity in response to carrageenin injection (2 mg) was significantly less in a group treated with YM-26734 (20 mg kg-1, i.v.) than in the control group (0.43 +/- 0.02 vs 0.59 +/- 0.03 g per cavity and 3.8 +/- 0.2 vs 4.9 +/- 0.3 x 10(7) cells per cavity, respectively; n = 5). 6. These results suggest that YM-26734 is a potent and competitive inhibitor of extracellular PLA2 with selectivity for group II PLA2, and that the inhibition of group II enzymes activity may cause the suppression of inflammatory responses to TPA and carrageenin.

NS-398, a novel non-steroidal anti-inflammatory drug with potent analgesic and antipyretic effects, which causes minimal stomach lesions

1. 1. NS-398 ( N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide) is a new non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic effects. 2. 2. The anti-inflammatory potency of NS-398 in rat carrageenin-induced edema was as potent as that of indomethacin and 8 times more potent than diclofenac. In rat adjuvant arthritis, NS-398 showed a therapeutic effect comparable to that seen with loxoprofen but less than that seen with indomethacin and diclofenac. 3. 3. The analgesic potency of NS-398 in rat adjuvant arthritic pain was much the same as that of indomethacin, and was about 3–5 times higher than that of diclofenac and loxoprofen. In the Randall-Selitto method in rats, NS-398 was 2–7 times as potent as loxoprofen, diclofenac and indomethacin. In acetic acid-induced writhing in mice, NS-398 was equipotent to indomethacin and diclofenac. 4. 4. In LPS-induced fever in rats, NS-398 was 1.5–4.5 times as potent as loxoprofen and indomethacin, but less potent than diclofenac. 5. 5. NS-398 produced little gastric ulceration in doses of up to 1000 mg/kg, while reference drugs produced distinct stomach lesions in doses of 10–30 mg/kg. 6. 6. NS-398 inhibited prostaglandin (PG) endoperoxide synthase from sheep seminal vesicle microsomes less potent than that of ibuprofen.

PI-specific phospholipase c “α” from sheep seminal vesicles is a proteolytic fragment of pi-plc σ

Phosphatidylinositide-specific phospholipase C enzymes (PLCs) catalyze the conversion of the phosphoinositides to biologically important signal transducing molecules. These enzymes may be grouped into “families” which share similar structures and modes of regulation. The existence of a structurally distinct family of PLC termed “α” has been recently called into question. In the current paper we show by immunoblotting experiments that PLC “α” from sheep seminal vesicles is recognized by monoclonal antibodies raised against the σ1 isoform of bovine brain PLC, and appears to be derived from a higher molecular weight band at 85 kDa. We also show that antibodies raised against PLC α efficiently immunoprecipitate the 85-kDa PLC σ1 isoform from bovine brain and Chinese hamster lung fibroblasts. These data provide strong evidence that the PLC a from sheep seminal vesicles is a proteolytic fragment of PLC σ1. Thus, there is still no conclusive evidence for a separate “α” class of PLC.