Floyd A. Green

Erythrocyte membrane sulfhydryl groups and Rh antingen activity

Abstract To study further the relation between Rh antigen activity and erythrocyte membrane sulfhydryl groups, the interaction of N-ethylmaleimide and iodoacetamide with human lyophilized erythrocyte membranes was quantitated by measuring the uptake of labeled reagent and liberation of iodile, respectively. N-ethylmaleimide reacts at room temperature with almost twice as many sulfhydryl groups as iodoacetamide and results in the gradual loss of Rh antigen activity over two hours. In contrast, no loss of antigen activity results from the reaction with iodoacetamide, even when the reaction waspushed by raising the sulfhydryl reagent to membrane concentration ratio, or when incubation was continued for eighteen hours. The spectrum of sulfhydryl group reactivity of iodoacetamide was included in the pattern of N-ethylmaleimide reactivity in that pretreatment of the membranes with N-ethylmaleimide almost completely suppresses iodide liberation on subsequent treatment with iodoacetamide. Urea in low concentrations does not increase the reactivity of sulfhydryl groups for iodoacetamide. In larger concentrations there is increased iodide liberation, but Rh antigen activity is lost without iodoacetamide. Parachloromercuribenzoate also results in loss of Rh antigen activity over two hours, but has a somewhat different spectrum of sulfhydryl reactivity in the erythrocyte membrane. Inorganic mercury results in a rapid, but reversible loss of Rh antigen activity at 0°C.

In vivo activation of an ω-6 oxygenase in human skin

To test the hypothesis that an epidermal fatty acid oxygenase is activated in vivo under physiologic conditions, surface lipids from normal human skin were analyzed for oxygenase products. With high-performance liquid chromatography on reversed-phase and straight-phase chiral columns and gas-liquid chromatography/mass spectrometry, these lipids were found to contain free 13-hydroxyoctadeca-9Z,11E-dienoic acid and 9-hydroxyoctadeca-10E,12Z-dienoic acid. The 13-hydroxyoctadecadienoic acid was present as a stereoisomeric mixture, with an average S/R ratio of 2.2, and exceeded the concentration of 9-hydroxyoctadecadienoic acid by a factor of 2. These observations and others indicate that the 13-hydroxyoctadecadienoic acid was derived mostly from an ω-6 oxygenase (probably 15-lipoxygenase) which is activated in vivo in normal skin.

In vivo generation of 5-lipoxygenase products in frogs and toads

Eicosanoid production by inflammatory cells which resulted from infection of the peritoneal cavity of Rana catesbeiana and Bufo americanus was studied after addition of exogenous arachidonic acid and for metabolites generated in vivo. From exogenous substrate, the cells of Rana catesbeiana produced substantial amounts of 5-hydroxyeicosatetraenoic acid, leukotriene B4, the non-enzymatic isomers of leukotriene B4 and leukotriene C4. From endogenous substrate, 5-hydroxyeicosatetraenoic acid and leukotriene B4 were produced. Cells from Bufo americanus produced leukotriene B4 and 5-hydroxyeicosatetraenoic acid, from both exogenous and endogenous substrate. These observations of in vivo eicosanoid production confirm the participation of 5-lipoxygenase activity in the inflammatory response to infection.

Interactions of a nonionic detergent. III. Further observations on hydrophobic interactions

Abstract Nonionic detergents of the Triton series are widely used as dispersing agents, but little is known about their capacity to interact with proteins and other molecules of biological interest. The critical micellar concentration of this detergent can be accurately measured taking advantage of an ultraviolet spectral shift during micelle formation, and this procedure was adapted to measure the amount of detergent bound to human and bovine serum albumin. In addition, the effects of the lower straight-chain alcohols on detergent micelle formation were investigated. Triton X-100 has an 8-carbon tertiary alkyl chain and 9–10 ethylene oxide units as the hydrophile. Triton X-102 has the same alkyl group, but 12–13 ethylene oxide units, and Triton N-101 has a 9-carbon alkyl chain and 9–10 ethylene oxide units. It was found that Triton N-101 bound almost as well to the albumins as the other two, but at a free concentration of approximately one-third of the others. The difference between the binding of Triton X-100 and Triton X-102 was only slight. This strongly suggests that hydrophobic interactions were most significant in the binding. Furthermore, low concentrations of n-butanol, but not secondary nor tertiary butanol, appeared to hydrophobically favor micelle formation of the Triton X-100 molecules. In comparable concentrations, methanol, ethanol and propanol had no such effect, tending rather to raise the critical micellar concentrations. This may have some relevance to the unique solubilizing role of n -butanol with retention of biological activities of proteins.

The phospholipid requirement for RH0(D) antigen activity: Mode of inactivation by phospholipases and of protection by anti-Rh0(D) antibody☆

Previous studies have suggested a membrane phospholipid requirement for Rho(D) antigen activity. Isolated erythrocyte membranes incubated with phospholipase A2 from both bee venom and porcine pancreas undergo loss of Rh antigen activity. The mode of attenuation of this antigen activity as indicated by double-reciprocal binding plots suggests substantial loss of sites accompanied by an apparently increased association constant. In the presence of anti-Rho(D), but not anti-A, bound to group A Rho(D)-positive membranes prior to hydrolysis, there is marked protection: almost complete preservation of sites at the expense of a decreased association constant. This pattern of protection is not seen with phospholipase C, which cleaves the polar headgroup in contrast to the A2-enzymes, which hydrolyze the fatty acid in the 2-position. Analysis of the products of digestion shows a trend to protection of bulk phospholipids of all major classes in the presence of bound specific antibody. The hydrophobic fatty acid chain may be the site with which the bound anti-Rho(D) antibody is in closest proximity.

Hypertonic cryohemolysis and the cytoskeletal system.

Hypertonic cryohemolysis is defined as the lysis of erythrocytes in a hypertonic environment when the temperature is lowered from above 15-18 degrees C below that temperature. This has been found to be a general phenomenon (that is, whether the solute is charged or not), to exhibit interesting temperature characteristics and to be preventable by agents such as valinomycin which tend to dissipate the concentration gradient across the cell membrane. As yet, no clear information is available to translate this phenomenon to the molecular level and to relate it to current structure/function concepts in the erythrocyte membrane. In this study, data are presented which would indicate on the basis of two entirely separate methodologies that the spectrin-actin cytoskeletal framework is involved in this phenomenon. The first of these methodologies is based on radiation-induced ablation of cryohemolysis and indicates that an intact macromolecular complex of an order of 16000 000 daltons is required for cryohemolysis with hypertonic NaCl. The second methodology is based on selective cross-linking of spectrin and actin in the agent diamide, which resulted in concentration-dependent suppression of cryohemolysis. Polyacrylamide gel electrophoresis of the erythrocyte from diamide-treated cells showed intense protein aggregation with loss of spectrin-actin and bands 4.1, 4.2. We conclude that the spectrin-actin cytoskeletal system possibly including its interaction with phospholipids is the key to the phenomenon of hypertonic cryohemolysis.

Divalent cation effects on the binding of human anti-phospholipid antibodies

Anti-phospholipid antibodies from sera of subjects with documented syphilis were measured as a result of their individual interactions with cardiolipin, phosphatidylserine and phosphatidic acid, each impregnated in nitrocellulose paper from chloroform solution, followed by reaction with a labelled second antibody. Binding curves generated by increasing the cardiolipin concentration over a standard area of nitrocellulose paper showed saturation. The presence of Ca2+ or Mg2+ during the antibody binding step resulted in a complex pattern of binding as a function of the cation concentration. When the extent of binding was normalized to percent of maximum bound, virtually super-impossible patterns were seen with different sera. These patterns were distinctive for both the phospholipid and the cation. The speculation is presented, albeit without any direct evidence, that the extent of antibody binding is sensitive to a variety of intermediate phospholipid phase structures which may be initiated by the presence of the specific divalent cation at a particular concentration.

The Rh antigen system and disaggregated human erythrocyte membranes.

Abstract Sodium deoxycholate was used to solubilize Rh(D) antigenic activity and other protein species of the human erythrocyte membrane. Detergent removal followed by the slow addition of Mg 2+ or Ca 2+ by dialysis, allowed the formation of membraneous structures from solubilized membrane protein. These membraneous structures contained serologically specific Rh(D) antigenic activity. Use of immobilized lectins from Phaseolus lunatus and Lens culinaris demonstrated that Rh(D) antigenic activity in the detergent-bound soluble membrane phase could be separated from A antigenic activity, from glucoprotein I and partly from glycoprotein II. Solubilization of Rh(D)-positive membrane- 14 C-anti-D antibody complexes followed by chromatography on agarose columns demonstrated that these complexes had a higher mol. wt than 14 C-IgG. This indicates Rh(D) antigen-anti-D antibody association in detergent and represents the first demonstration of this antigen activity in an all-soluble system.

Saturability of esterification pathways of major monohydroxyeicosatetraenoic acids in rat basophilic leukemia cells

The principal monohydroxyeicosatetraenoic acids (HETEs), 5-, 12-, and 15-HETE, which can be produced by rat basophilic leukemia (RBL-1) cells, are also esterified by these cells. Exogenously added 5-, 12-, and 15-HETE were rapidly incorporated as esters in RBL cells, reaching plateau levels within 25 min. In incubations in culture medium with protein added, all three HETEs were essentially completely metabolized within 24 h. 5-HETE was esterified more rapidly and to a greater extent than 12-HETE or 15-HETE when these were incubated together with RBL cells, indicating some degree of selectivity in the esterification pathways. When arachidonic acid (AA) was incubated in increasing concentrations with constant concentrations of 15-HETE and RBL cells, the free 15-HETE concentration increased and esterified 15-HETE concentration decreased markedly at AA: 15-HETE molar ratios above 9. 15-HETE esterification in RBL cells was also markedly inhibited by the polyunsaturated fatty acids, eicosatetraynoic and eicosapentanoic acids, but not by oleic or linoleic acids. In separate experiments with unlabeled and radiolabeled substrates, the extent of incorporation of esterified HETE in RBL cells decreased at higher concentrations of 15-HETE and AA, which showed that the pathway was saturable. The shapes of the curves for these fatty acid inhibitors suggest a concentration-dependent two compartment pathway of esterification. These data indicate that the HETEs and other 20 carbon fatty acid substrates probably compete for activity of a specific arachidonyl-CoA synthetase, which is the first and rate-limiting step for esterification of arachidonic acid by many human cells. Esterified 15-HETE was found to be predominantly in the phosphatidylethanolamine fraction of RBL cell lipids.

Lipoxygenase products in inflammatory synovial fluids and other exudates.

Forty six synovial fluid samples from 42 patients with inflammatory joint disease were analysed by reversed phase high performance liquid chromatography to determine 5-lipoxygenase products, specifically dihydroxyeicosatetraenoic acids (diHETEs). Twenty eight per cent of the fluids which were assayed had one or more products of 5-lipoxygenase activation. Seven fluids contained leukotriene B4 (0.1-28.1 ng/ml); three fluids had low concentrations of 20 carboxy/hydroxy-leukotriene B4 (0.01-0.05 ng/ml); three samples had leukotriene B4 isomers (1.5-2.4 ng/ml); and four fluids contained 5,15-diHETE (2.3-16.4 ng/ml). There was a poor correlation between synovial fluid white blood cell counts and evidence of 5-lipoxygenase activation. Several fluids contained unidentified compounds with spectra similar in shape to that of trienes, but the lambda max values of these unidentified compounds were different from those of known leukotrienes. A septic peritoneal exudate and a septic pleural fluid had concentrations of leukotriene B4 and leukotriene B4 isomers and metabolites in a range similar to those found in synovial fluids.