Crotalus Adamanteus Research Articles

The role of phospholipase A2 in interleukin-1 alpha-mediated inhibition of mineralization of the osteoid formed by fetal rat calvaria cells in vitro.

Interleukin-1 (IL-1) may be an important mediator of bone remodeling, since it is a potent stimulator of bone resorption and has biphasic effects on bone formation. Continuous exposure of fetal rat calvaria (RC) cells to IL-1 alpha or IL-1 beta results in a dose-dependent inhibition of both bone nodule formation and mineralization of the organic matrix. In this study, the effects of recombinant human IL-1 alpha on the mineralization process were examined by the addition of IL-1 alpha late in the culture period, after osteoid nodules had formed and when they were induced to mineralize by the addition of organic phosphate. By means of a quantitative 45calcium radiolabeling assay, it was shown that short-duration exposures of fully-formed bone nodules to IL-1 alpha also inhibited mineralization, and that the duration of treatment directly correlated with the degree of inhibition. Because our earlier studies had demonstrated that IL-1 stimulated the release of PLA2 and PGE2 from RC cells, the effects of PLA2 and of inhibition of PGE2 synthesis on mineralization were investigated. Exogenous Naja naja group I PLA2 had little effect on the mineralization of bone nodules; however, Crotalus adamanteus group II PLA2 inhibited mineralization at concentrations similar to those found in the media from IL-1 alpha-treated cultures. Although PLA2 is thought to stimulate PGE2 synthesis by releasing arachidonic acid from membrane phospholipids, PGE2 release by RC cells accounted for only part of the IL-1 alpha-mediated inhibition, suggesting the presence of other mechanisms of exogenous PLA2 action in inhibiting mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical study of phospholipase A2 in boyine prostate

Approximately 1,100-fold purified phospholipase A2 (PLA2) from bovine prostate was injected into rabbit to prepare polyclonal antibodies. Antibodies produced showed specific immunoprecipitation only with the purified enzyme, as well as with homogenate of bovine prostatic tissue. By Western blot analysis or by immunodiffusion test, no cross-reactivity with PLA2 purified from human seminal plasma, bovine pancreas, Crotalus adamanteus venom, or with partially purified PLA2 from bovine seminal vesicle fluid or Cowper's glands was observed. Using the indirect peroxidase technique, PLA2 was localized in the cytoplasm of bovine prostatic epithelial cells. By immunogold microscopy, this enzyme was directly visualized inside the lysosomes, as well as in the endoplasmic reticulum of the glandular epithelial cells. Enzyme activity was localized in two principal subcellular sites: the mitochondria and lysosome-enriched fraction, and in the microsomal fraction.

Evaluation of four different immunogens for the production of snake antivenoms

Four different immunogens were used to produce polyvalent antivenom in rabbits to the venoms of Bothrops atrox, Crotalus atrox, Crotalus adamanteus and Crotalus durissus terrificus. The immunogens were: (1) unfractionated mixture of the four crude venoms, and three fractions of the mixture as follows, (2) HPLC gel filtration high (> 50,000) mol. wt fraction, (3) HPLC gel filtration medium (14,000–50,000) mol. wt fraction, and (4) HPLC gel filtration low (< 14,000) mol. wt fraction. The resultant immune sera were compared with commercial antivenom (Wyeth, polyvalent Crotalidae) for total IgG content, ELISA reactivities, patterns of Western blots and ability to neutralize lethal and local hemorrhagic activities of the four venoms. The results indicate that the rabbit antivenoms had significantly higher ELISA reactivity and blotting signals than Wyeth antivenom. However, neither ELISA nor Western blotting signals correlated with the ability of the antivenoms to neutralize the lethal or hemorrhagic activities of the venoms. The protective ability of the antivenoms varied considerably. In general, antivenoms generated by using fractionated venoms as immunogens exhibited greater protective ability than antivenom produced by using the mixture of four venoms as immunogen. Some of the antivenoms provided greater or comparable protective ability for certain venoms when compared to Wyeth antivenom. It appears that the use of certain venom fractions as immunogens is a promising alternative for production of effective antivenoms.

Citrate is an endogenous inhibitor of snake venom enzymes by metal-ion chelation

Citrate levels in selected snake venoms were determined by an enzymatic assay coupled to NADP + reduction. Citrate concentrations in different viper venoms ( n = 5) varied from 95 to 150 mM, in crotalids ( n = 3) from 63 to 142 mM, and in elapids ( n = 4) from 17 to 163 mM. In Bothrops asper venom Ca 2+-ion concentrations varied from 2.5 to 3.6 mM, suggesting that the high relative citrate levels may serve to chelate endogenous divalent metal cations, thereby inactivating divalent cation requiring enzymes. Control experiments with B. asper phospholipase A 2 MIII in the presence of 2.5 mM Ca 2+, showed that the enzyme is completely inhibited by 20 mM citrate. Crotalus adamanteus 5′-nucleotidase and phosphodiesterase are also inhibited 100 and 75%, respectively, by 100 mM citrate. By forming complexes with divalent metal ions, citrate markedly reduces the activities of selected enzymes in snake venoms. Secretion of high concentrations of citrate may represent an important mechanism by which snakes protect themselves against the toxic effects of their own venoms.

Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine

Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report that sphingosine directly inhibits phospholipases A 2 and D. Sphingosine inhibits Ca 2+-dependent phospholipases A 2 from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrophil in a dose-dependent manner with IC 50, values ranging from 5–40 μM using [1- 14C]oleate-labelled autoclayed E. coli (20 μM) as substrate. Inhibition is comparable using the same concentrations (20 μM) of [1- 14C]oleate-labelled C. elbicans or E. coli, or aqueous dispersions of 1-acyl-2-[1- 14C]linolcoylglycerophosphoethanolamine or -choline. Sphinganine and stearylamine are as inhibitory as sphingosine; monoolein is less inhibitory (IC 50 = 70 μM), while octylamine, N-acetylsphingosine, sphingomyelin and ceramide have no effect. inhibition is relieved by increasing concentrations of substrate phospholipid. The molar ratio of sphingosine to phospholipid required for 50% inhibition ranges from 0.5 to 1.0 with 2–100 μM E. coli phospholipid. In contrast, sphingosine has a biphasic effect on the hydrolysis of E. coli by S. chromofuscus phospholipase D, concentrations ⩽25 μM stimulate activity while concentrations 25 μM are inhibitory. Addition of Triton X-100 climinates both the stimulatory and inhibitory effects of sphingosine on phospholipase D activity.

Transformation of N epsilon-CBZ-L-lysine to CBZ-L-oxylysine using L-amino acid oxidase from Providencia alcalifaciens and L-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus.

Biotransformations were developed to oxidize N epsilon-carbobenzoxy(CBZ)-L-lysine and to reduce the product keto acid to L-CBZ-oxylysine. Lysyl oxidase (L-lysine: O2 oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for L-lysine and had very low activity with N epsilon-substituted derivatives. L-Amino acid oxidase (L-amino acid: O2 oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with L-lysine but high activity with N epsilon-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-L-lysine. L-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N epsilon-acetyl-, trifluoroacetyl-, formyl- and CBZ-L-lysine but was inactive with the products from oxidation of L-lysine, L-lysine methyl ester, L-lysine ethyl ester or N epsilon-t-BOC-L-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H2O2:H2O2 oxidoreductase EC 1.11.1.6). N epsilon-CBZ-L-Lysine was converted to CBZ-L-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using L-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions.

Reversibility of the activation of soluble phospholipase A 2 on lipid bilayers: Implications for the activation mechanism

The time-courses of hydrolysis of large vesicles of dipalmitoylphosphatidylcholine were compared using four species of phospholipase A 2 ( Agkistrodon piscivorus piscivorus, Crotalus adamanteus and Naja naja venoms and porcine pancreatic). In all four cases, the hydrolysis rate suddenly increases 10 to 100-fold at the time (τ) when a specific mole fraction of reaction products has accumulated. The intrinsic fluorescence emission of the three venom enzymes also increases suddenly at time τ. Both the activation and the fluorescence change are reversible with a half-time of about 50 s for the activity and 2 to 6 s for the fluorescence. These reversal rates and the vesicle concentration dependence of τ are considered for monomer and dimer enzyme activation models. Apparently, at least three states of the enzyme exist beyond the initial unbound state: (1) inactive and bound, (2) inactive with high fluorescence and (3) active. The dimer model already contains the necessary number of states but requires that the activation rate be much lower than the reversal rate to account for the vesicle concentration dependence of τ. Success of the monomer model requires an enzyme state additional to those proposed previously. Although these results do not exclude either the monomer or dimer models conclusively, they do impose important constraints on each model.

Peroxidation and phospholipase A 2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine

The relationship between lipid peroxidation and phospholipase A 2 (PLA 2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixturesof these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced vesicles with cumene hydroperoxide and hematin at 37°C. Determinationsof the extent of peroxidation by means of diene conjugate conjugate derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA 2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA 2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA 2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA 2 PLA 2.

An immunohistochemical study of phospholipase A 2 in peripheral nerve during Wallerian degeneration

Antiserum raised against purified Crotalus adamanteus venom phospholipase A 2 (PLA 2) was used to localise the enzyme in normal and crushed sciatic nerve to determine the effect of nerve trauma on PLA 2 distribution in the PNS. The tissue was Bouin's fixed, and stained immunohistochemically using the Vectastain ABC technique. Anti-PLA 2 antiserum showed weak reactivity with normal rat myelin, while only ‘resident’ macrophages stained in normal mouse sciatic nerve. Post-crush, in the mouse, myelin staining was localised close to the crush site, with strong lysosomal staining within both ‘resident’ and invading haematogenously derived macrophages. In the rat, myelin sheats stained strongly throughout the nerve very soon after crush, but no macrophages were detected using these antisera. Paranodal myelin and some Schmidt-Lanterman incisures were the first regions of the sheath to show an increase in PLA 2 staining post-crush, areas where the first ultrastructural changes occur in myelin after trauma, with staining of compact myelin closely following. Our findings support the idea that PLA 2 may be involved in the early process of myelin breakdown seen in Wallerian degeneration.

Citrate is a major component of snake venoms

Citrate has been identified as a major component of snake venoms by gas liquid chromatography and mass spectrometry. The venoms of Bothrops asper, Crotalus atrox, Crotalus viridis viridis, Crotalus adamanteus, Sistrurus miliarius barbouri, Crotalus horridus horridus, Agkistrodon contortrix mokasen, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus contain citrate at concentration levels which can serve as effective buffers. Calcium, magnesium, zinc, iron, sodium and potassium salts of citrate would be constituents of venom.

Interleukin-1α stimulates the release of prostaglandin E2 and phospholipase A2 from fetal rat calvarial cells in vitro: Relationship to bone nodule formation

We have shown previously that interleukin-1 (IL-1) has biphasic effects on the formation of bone nodules in long-term cultures of fetal rat calvarial (RC) cells (Ellies and Aubin, Cytokine 2:430-437, 1990). To determine the role of arachidonic acid metabolism in this process, we have examined the release of prostaglandin E2 (PGE2) and phospholipase A2 (PLA2) from RC cells under conditions that allowed concomitant analysis of the formation of bone nodules. Recombinant human IL-1 alpha (rhIL-1 alpha) stimulated PGE2 and PLA2 release in a time- and dose-dependent manner. PGE2 release was highest in preconfluent cultures (days 1-6) and was stimulated up to 8.5-fold in response to 50 U/ml of rhIL-1 alpha. In contrast, extracellular PLA2 activity was maximal in postconfluent cultures, with 50 U/ml of rhIL-1 alpha causing a 20-fold increase by day 15. PLA2 release by RC cells was not significantly affected by PGE2, the glucocorticoid dexamethasone, or the cyclooxygenase inhibitor indomethacin. Indomethacin partially blocked the inhibition of bone nodule formation caused by rhIL-1 alpha, and exogenous PGE2 reversed this effect. Addition of group I PLA2 from Naja naja venom to RC cells had no effect on bone nodule development; however, group II PLA2 from Crotalus adamanteus venom inhibited the formation of bone nodules in a dose range similar to that induced by rhIL-1 alpha. These results indicate that PGE2 release does not have a direct temporal correlation with increases in PLA2 activity. In addition, the data show that only part of the inhibition of bone formation seen with rhIL-1 alpha is mediated by PGE2 and suggest that extracellular PLA2 also accounts for part of the inhibition.

Identification of a 15-kilodalton surface glycoprotein on sporozoites of Cryptosporidium parvum

An immunoglobulin A monoclonal antibody (MAb5C3) was developed against a 15-kDa surface glycoprotein (GP15) of Cryptosporidium parvum sporozoites. Indirect immunofluorescence and colloidal gold immunoelectron microscopy revealed that the antibody reacted with both the sporozoite and merozoite surface plasma membranes. On Western immunoblots, MAb5C3 binding was found to be strongly inhibited when 200 mM N-acetylglucosamine was used as a competing sugar. N-Acetylgalactosamine inhibited binding of the antibody only slightly, whereas glucose, mannose, and galactose failed to inhibit binding. MAb5C3 was found to recognize a similar 15-kDa epitope associated with a Cryptosporidium sp. isolated from guinea pigs. However, MAb5C3 failed to react with any proteins or glycoproteins associated with C. baileyi from chickens, Cryptosporidium sp. (= bovine C. muris) from cattle, C. serpentis from a rat snake, bradyzoites of Besnoitia darlingi from an opossum, sporozoite/oocyst extracts of Caryospora bigenetica from an eastern diamondback rattlesnake, sporozoites of Eimeria nieschulzi and E. papillata from rats and mice, or tachyzoites of Toxoplasma gondii (RH strain). When hybridoma supernatants containing MAb5C3 were administered orally to suckling mice experimentally infected with C. parvum, a 75% reduction in developmental stages was seen histologically at 72 h postinfection and a 67.5% reduction in mean oocyst output was found at 6 days postinfection.

Suicide-inhibitory bifunctionally linked substrates (SIBLINKS) as phospholipase A2 inhibitors. Mechanistic implications.

Novel phospholipids that function as mechanism-based inhibitors for phospholipase A2 (PLA2) are described. PLA2-catalyzed hydrolysis of the sn-2 ester of these suicide-inhibitory bifunctionally linked substrates (SIBLINKS) followed by a cyclization reaction generates a cyclic anhydride at the active site of the enzyme which leads to inhibition. Structure/activity relationships for the SIBLINKS substituents in the sn-1 and sn-2 position are delineated. Time courses and efficiency of SIBLINKS inhibition are reported and compared for extracellular PLA2s obtained from Naja naja naja, porcine pancreas, bee venom, Crotalus atrox and Crotalus adamanteus. SIBLINKS-inhibited PLA2s cannot process either monomeric or micellar substrates consistent with inhibition at the catalytic site. Some SIBLINKS efficiently inactivate 1 mol of N. naja naja and C. adamanteus PLA2/6-10 mol of SIBLINKS hydrolyzed. Inhibition of N. naja naja PLA2 can be reversed by hydroxylamine, suggesting that a tyrosine residue is acylated.

Amino acid sequence of a myotoxin from venom of the eastern diamondback rattlesnake (Crotalus adamanteus)

Y. Samejima, Y. Aoki and D. Mebs. Amino acid sequence of a myotoxin from venom of the eastern diamondback rattlesnake ( Crotalus adamanteus). Toxicon 29, 461–468, 1991.—The complete amino acid sequence of a myotoxin isolated from Crotalus adamanteus venom (CAM-toxin) was determined. The total number of amino acid residues was 45, giving a mol. wt of 5202. The amino acid sequence was compared with those of previously determined Crotalus myotoxins. The CAM-toxin shows a surprisingly close relationship to those of peptide c ( C. v. helleri), myotoxin a ( C. v. viridis), crotamine ( C. d. terrificus), myotoxin I and II ( C. v. concolor) with homologies of approximately 98, 83, 90, 95 and 91%, respectively. The hydropathy profile of the CAM-toxin is almost identical to peptide c and myotoxin I, as can be assumed from the sequence homology. Despite the absence of any close sequence homology with the myotoxic phospholipase A 2 enzymes, CAM-toxin contains a similar cationic myotoxic region at residues 2–10 as found in the other five myotoxic peptides.

Behaviour of phospholipase modified-HDL towards cultured hepatocytes. II. Increased cell cholesterol storage and bile acid synthesis

Human total HDL (hydrated density 1.070–1.210), HDL 2 (1.070–1.125), HDL 3 (1.125–1.210) or HDL separated by heparin affinity chromatography were treated with or without purified phospholipase A 2 from Crotalus adamanteus. Control and treated HDL were reisolated and were then incubated with cultured hepatocytes. 1. Mass measurements evidenced a time-dependent cholesterol enrichment in hepatocytes cultured in the absence of lipoproteins. Addition of HDL 2 still enhanced by 25% the cell cholesterol content and down-regulated endogenous sterol synthesis in similar proportions. Conversely, HDL 3 slightly decreased the amount of free cholesterol in hepatocytes (−12%). 2. Incubations with phospholipase A 2-treated HDL resulted in a 35%–50% increase of both the cellular cholesterol esterification and the cholesterylester accumulation, when compared to cells cultured in the presence of control-HDL. This effect was observed with HDL 2, HDL 3 and combining the data with all subfractions. 3. Cultured hepatocytes secreted cholic and β-muricholic acids as major bile acids and HDL 2 showed a tendency to stimulate their secretion. Phospholipase treatment of HDL again induced an increased production by hepatocytes of those two bile acids. Thus, whereas HDL 2 and HDL 3 display different behaviours with respect to cell cholesterol content, neosynthesis and bile acid secretion, their modifications by phospholipases always orientate the cell sterol metabolism in the same direction: increased cholesterylester accumulation and bile acid production.

Neutralization of kinin-releasing enzymes of crotalid venoms by monospecific and polyspecific antivenoms

G. S. Bailey, A. Al-Joufi, S. Rawat and D. C. Smith. The neutralization of kinin-releasing enzymes of crotalid venoms by monospecific and polyspecific antivenoms. Toxicon 29, 777–781, 1991.—The amounts of kinin-releasing enzymes in the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus scutulatus and Agkistrodon piscivorus piscivorus were measured by determining the amounts of kinin released from a sheep kininogen substrate by means of a specific radioimmunoassay. Four monospecific and two commercial polyspecific antivenom IgG samples were tested for their ability to reduce the kinin-releasing activities of the four crotalid venoms measured in vitro. All of the antivenom IgG samples were able to neutralize venom kininogenase activity to varying extents. One of the commercial polyspecific antivenoms was of equal or higher potency than the corresponding monospecific antivenoms for three of the venoms tested, indicating a high degree of cross-neutralization. F(ab’) 2 and especially Fab fragments of that polyspecific antivenom IgG were also effective in reducing the kinin-releasing activities of the four crotalid venoms.

A new affinity adsorbent for the purification of phospholipases A 1 and A 2 from animal venoms

Dimethyl- dl-2,3-distearoyloxypropyl-2′-hydroxy-ethylammonium (Rosenthal's inhibitor) was coupled to carboxyhexyl-Sepharose 4B, through carbodiimide chemistry. Phospholipase A 2 from Heloderma horridum horridum and Crotalus adamanteus bind to the immobilized ligand in the presence of Ca 2+ and can be easily eluted under acidic conditions or in the presence of a chelating agent, respectively. This affinity media proved to be effective also in the purification of a Ca 2+-independent phospholipase A 1 from vespid venom.

Regional differences in content of small basic peptide toxins in the venoms of Crotalus adamanteus and Crotalus horridus

1. 1. Reverse-phase HPLC and organic solvents were used to isolate small basic peptide (SBP) toxins from the venoms of Crotalus adamanteus, C. durissus terrificus, C. horridus, C. scutulatus scutulatus, C, viridis concolor, C. viridis helleri and C. viridis viridis. 2. 2. Acid-DEP analyses indicated a high degree of toxin purity which was obtained with a single HPLC run. 3. 3. The combined results of HPLC, immunodiffusion and electrophoresis analyses of venoms from different geographical regions indicate that the SBP toxin content in the venoms of Crotalus adamanteus, Crotalus horridus, Crotalus scutulatus and Crotalus viridis may vary regionally.

Purification and characterization of a soluble phospholipase A 2 from guinea pig lung

Guinea pig lung cytosolic phospholipase A 2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A 2 hydrolyzed phosphatidylcholine and phosphatidylethanol-amine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A 2 under conditions in which Crotalus adamanteus phospholipase A 2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A 2 in a concentration-dependent manner ( IC 50 = 2 μM). Antibodies prepared against porcine pancreatic phospholipase A 2 specifically immunoprecipitated guinea pig lung phospholipase A 2 suggesting that the major phospholipase A 2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A 2 in agreement with the biochemical properties of the enzyme.

Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins

Human HDL subfractions (HDL 2, HDL 3, or HDL separated by heparin affinity chromatography) were labelled either on their apolipoprotein moiety with 125I or on their sterols: unesterified [ 14C]cholesterol and [ 3H]cholesteryl linoleyl ether, a non-hydrolysable analog of esterified cholesterol. HDL subfractions were then treated with or without phospholipase A 2 from Crotalus adamanteus in presence of albumin leading to a 72–82% phosphatidylcholine degradation. Control and treated HDL were reisolated and then addressed to cultured rat hepatocytes. (A) During incubations, unesterified [ 14C]cholesterol from HDL 3 readily appeared in hepatocytes. The specific uptake of HDL esterified cholesterol calculated from [ 3H]cholesteryl ether was 2–4-times less important. Uptake of HDL cholesterol tended to saturate at 150–200 μg/ml HDL protein. A prior phospholipase treatment of HDL 3 stimulated by 2–5-fold the uptake of [ 3H]cholesteryl ether, whereas the transfer of free [ 14C]cholesterol was minimally increased. The uptake of 3H/ 14C-labelled sterols from HDL 2 was 2–3-times higher than from HDL 3. (B) Parallel experiments were conducted with 125I-labelled HDL subfractions. At 37°C, the specific uptake and degradation of HDL 3 125I-apolipoprotein were about 2-fold enhanced following treatment of HDL 3 with phospholipase A 2. Uptakes of apolipoprotein and of esterified cholesterol were compared, indicating a preferential delivery of the sterol over apoprotein (×5). The dissociation was still more pronounced with phospholipase-treated HDL 3. Competition experiments showed that 12-times more unlabelled HDL 3 were required to half reduce the uptake of HDL 3 [ 3H]cholesteryl ether than to impede similarly the HDL 125I-apolipoprotein recovered in cells. Uptake of 125I-labelled apolipoprotein from HDL 2 was quantitatively comparable to that from HDL 3. (C) Binding of 125I-HDL subfractions was followed at 4°C. A specific binding was observed for HDL 2 and HDL 3, although kinetic parameters were quite different ( K D of 9 and 25 μg/ml, respectively). Following phospholipolysis, both the specific and non-specific contributions to total binding were increased. Hence, hepatocytes take up more 125I-labelled apolipoprotein and 3H/ 14C-labelled sterols from lipolysed HDL than from unmodified particles. This is associated to changes in the binding characteristics.