Donald J. Hanahan

The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes.

Abstract The effects of the ionic strength and pH of the hemolyzing solution on the hemoglobin content of human erythrocyte ghosts were studied in phosphate buffers and found to have a pronounced influence upon hemoglobin binding in the ghosts. Buffer concentrations between 10 and 20 ideal milliosmolar (imOsm), at pH values 5.8 – 8.0, resulted in maximum hemoglobin removal from ghosts. The pH optimum for hemoglobin binding to ghosts was between 5.8 and 5.9 in a 20 imOsm buffer. The influence of these variables suggest an electrophysical interaction of hemoglobin with membrane constituents. This study provides a basis for comparison of existing methods for ghost preparation, as well as a means for prediction of the conditions required for preparation of ghosts containing any desired amount of hemoglobin. Conditions were found that allowed the preparation of hemoglobin-free ghosts by single-stage hemolysis and washing. Hemoglobin-free ghosts were prepared in 20 imOsm phosphate buffer at pH 7.4. Essentially all the lipid was recovered in the ghosts, but non-hemoglobin nitrogen-containing substances were lost. The pyridine hemochromogen method for hemoglobin determination was adapted for the measurement of very small quantities of hemoglobin through use of the Soret band (418 mμ) for absorbancy measurements.

Enzyme and hemoglobin retention in human erythrocyte stroma

Abstract Retention of aldolase (ketose-1-phosphate aldehyde-lyase, EC 4.1.2.7) and glyceraldehyde phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) by human erythrocyte stroma has been shown to be dependent upon pH and osmotic strength. Limited changes in these parameters have no effect, however, upon the complete retention of acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) by the stroma. Stroma which have been prepared almost completely free of hemoglobin, aldolase, and glyceraldehyde phosphate dehydrogenase can still interact with hemoglobin, suggesting that irreversible changes need not be involved in removal of protein from the stroma.

Chemical synthesis of 1-O-(D)- and 3-O-(L)-glyceryl monoethers, diethers and derivatives: Glycerides, monoester phospholipids and diether phospholipids

Abstract 1. 1. A convenient route to the synthesis of 1- O -glyceryl ether from 3- O -glyceryl ether is reported. This technique, which is based on the procedure of Lands and Zschocke 35 for conversion of 3- O -benzylglycerol to 1- O -benzylglycerol, utilized either 3- O -hexadecylglycerol or 3- O -octadecylglycerol as the starting material. The ether was converted to its ditosylate and this latter group was displaced with freshly fused potassium acetate in anhydrous ethanol. Alkaline hydrolysis yielded the desired 1- O -alkyl glycerol, with an equal but opposite optical rotation to the starting material, in good yields. 2. 2. 1- O - cis -9-Octadecenyl-2- O -octadecanoylglycerol (2-stearoyl selachyl al-cohol) was synthesized by borate replacement of 1- O - cis -9-octadecenyl-2- O -octade-canoyl-3- O -tritylglycerol in triethyl borate and boric acid, followed by column chromatography on neutral silicic acid. Catalytic amounts of HClO 4 isomerizes 2-stearoyl selachyl alcohol completely to its positional isomer 3-stearoyl selachyl alcohol. Acylation of 1- O -cis-9-octadecenylglycerol in the presence of up to 2 M excess of acyl chloride, was found to occur preferentially at the primary hydroxyl group forming 3-stearoyl selachyl alcohol and the diacyl derivatives (dipalmitoyl, dioleoyl and distearoyl selachyl alcohol) formed in good yield only when a 4–6 M excess of acyl chloride was used. The properties of the synthetic diacyl-glyceryl derivatives correlated closely with the natural diacyl-glyceryl monoether isolated from dog fish liver oil. 3. 3. 1,2-Di- O - cis -9-octadecenylglycerol which was synthesized by acid hydrolysis of its trityl derivative was converted to the corresponding phosphorylethanolamine and phosphorylcholine derivatives; 2- and 3-stearoyl selachyl alcohol were converted to their phosphoryl ethanolamine and phosphorylcholine derivatives. The physical properties of the monoethers, monoacyl monoethers, diethers and their corresponding phospholipids are tabulated and discussed. Comparable results in properties between the synthetic 1- O - cis -9-octadecenyl-2- O -octadecanoyl-3-glycerophosphorylethanolamine and the natural monoether monoacyl glycerophosphorylethanolamine isolated from bovine erythrocytes were found.

Influence of Surface Charge of Phospholipids on their Clot-Promoting Activity.

Summary Electrophoretic mobilities and thromboplastic activities of emulsions of mixtures of phospholipids derived from beef brain and egg yolk were determined. The studies included mixtures of varying amounts of phosphatidyl serine in lecithin, phosphatidyl serine in phosphatidyl ethanolamine, and phosphatidic acid in lecithin. It was concluded that the manifestation of “thromboplastic activity”by a particular phospholipid emulsion will depend largely upon the surface charge of the particles. The possibility of other factors involved was not excluded.

Phospholipid metabolism in kidney. I. Isolation and identification of lipids of rabbit kidney

Abstract Separation and isolation of the individual neutral and phospholipids of rabbit kidney cortex and medulla were achieved by chromatographic means. Results of the detailed analysis of the lipid classes thus obtained are presented. The kidney was found to contain large amounts of cholesterol among the neutral lipids and chiefly phosphatidyl choline and phosphatidyl ethanolamine in the phospholipids. Considerable amounts of the vinyl-ether analogue of phosphatidyl ethanolamine were also identified. An unusual inositol phospholipid having a high ester to phosphorus ratio was found to be present. Studies of fatty acid composition were carried out on the lipid fractions. A certain degree of specificity was noted in the positioning of fatty acids in lecithin and triglyceride, and several differences were found between the lipids of cortex and medulla.

The preparation and properties of a stable factory V from bovine plasma

Abstract 1. 1. Examination of highly purified bovine factor V revealed that under controlled conditions it is extremely stable even at 37° and therefore can be utilized in studies of its role in blood coagulation. The main requirement for stability in solution is simply that the total protein concentration be at least 0.1 mg/ml. More dilute solutions of factor V can be stabilized by addition of crystalline bovine plasma albumin. 2. 2. The effects of Ca2+ on the factor V preparation have been investigated and the possible role of Ca2+ in stabilizing factor V in plasma is discussed. 3. 3. On addition of a small amount of thrombin an immediate increase in activity measurable in the factor V assay was observed; at the same time, thrombin caused an increased retention of factor V during chromatography on Sephadex G-200 columns. The mechanism of this effect has not been firmly established. 4. 4. The preparation of factor V from bovine plasma in good yield and in a high state of purity is described. This technique, which is a modification of a previously reported procedure (see ref. 4), involves five steps; BaSO4 adsorption, chromatography of the “adsorbed” plasma on TEAE-cellulose and on cellulose phosphate, rechromatography on cellulose phosphate and precipitation with CaCl2. Factor V is obtained in 25–40% yield and 6000-10 000-fold purified. 5. 5. Factor V prepared as described can react with highly purified activated factor X, phospholipid and Ca2+ generating a potent prothrombinase activity.

Some lipid-protein interactions involved in prothrombin activation.

Abstract 1. 1. Bovine plasma prothrombin forms a macromolecular complex with an equimolar mixture of phosphatidylserine and phosphatidylcholine in aqueous dispersion. Complex formation, as detected by gel filtration on columns of Sephadex G-200, occurs only in the presence of an optimal concentration of Ca 2+ . 2. 2. Thrombin, bovine plasma albumin and an α 1 -acid glycoprotein are not adsorbed by the lipid particles either in the presence or absence of Ca 2+ . 3. 3. At—1°, prothrombin becomes attached to the particulate prothrombin activator complex consisting of activated factor X, factor V, phosphatidylserine/phosphatidylcholine mixture and Ca 2+ . At 37° in this system, prothrombin is converted to thrombin which itself is not adsorbed by the lipoprotein complex. 4. 4. The kinetics of prothrombin activation are consistent with a model involving catalysis of the reaction at the lipid-water interface and subsequent release of thrombin into the aqueous phase. Such a mechanism would favour the forward reaction by removing the reaction products from the site of their formation, at the same time creating sites for the renewed adsorption of prothrombin.

Metabolism of α-glyceryl ethers by Tetrahymena pyriformis II. Properties of a cleavage system in vitro

Abstract The biocleavage of glyceryl ethers (especially of chimyl alcohol) in Tetrahymena pyriformis W cell-free preparations was investigated. Tetrahymena crude homogenates or subcellular fractions are 45–70 times more active than has been reported for comparable rat liver preparations. The system is similar to cell-free liver preparations in that the reaction requires molecular O2, NAD+ and an NADPH-generating system as cofactors for maximal activity. However, it was shown that neither a pteridine nor any other low-molecular weight compound soluble in the microsomal supernatant is involved in this reaction. Instead, the enzyme activity is distributed as an independent entity during subcellar fractionation, being principally recovered in the pellet resulting from centrifugation of the microsomal supernatant at 250000 × g. The major product of the glyceryl-ether breakdown was identified as fatty acid produced from the hydrocarbon side chain of the chimyl-alcohol molecule. Two other products were detected, one migrating as fatty aldehyde on thin-layer chromatography and another as fatty alcohol. The formation of the aldehyde-like compound does not seem to precede the formation of the product labeled as “fatty alcohol”, and no particular accumulation of radioactive fatty aldehyde was observed by omitting NAD+ from the reaction mixture or by including in it a bulk quantity of unlabeled palmitaldehyde. It was demonstrated that this ability of Tetrahymena to degrade glyceryl ethers in an in vitro system is not inducible and no significant changes in this enzyme specific activity were observed at various stages of Tetrahymena growth. Finally, the present system was found to possess comparable ability to cleave glyceryl-vinylic and glycol ethers, but not glyceryl-ether phosphatides.

Metabolism of α-glyceryl ethers by Tetrahymena pyriformis I. Characteristics of the in vivo degradation system

Abstract 1. 1. Tetrahymena pyriformis W absorbs, within 30 min, over 90% of the [ 3 H]-chimyl alcohol added to the growth medium as an aqueous dispersion in concentrations up to 1.5–2.0 μmoles per 10 7 cells. After the initial period, a small proportion of the absorbed glyceryl ether remains in the free form and continues to be incorporated slowly into the lipids. The major part of absorbed chimyl alcohol is rapidly incorporated (either intact, or after production of fatty acids through cleavage of the ether bond) into the cellular lipids, whose composition is thereby significantly altered. As ths amount of administered chimyl alcohol increases, the content of glyceryl-ether containing phospholipid reaches maximal levels and abnormally high amounts of triglyceride accumulate. 2. 2. The rate of glyceryl-ether cleavage is largely proportional to the amount of chimyl alcohol added, being very pronounced at high concentrations of added substrate. However, it has been shown that this ability of Tetrahymena to degrade glyceryl ethers is not due to the action of induced enzymes. 3. 3. The possibility of the formation of two substrate pools upon glyceryl-ether absorption by the cells, as suggested by the radioactivity patterns and the time-course study of chimyl-alcohol incorporation into the lipids, was further investigated by blocking vacuole formation with dinitrophenol. This mechanism of glyceryl-ether utilization by Tetrahymena cells is discussed in detail.

The biosynthesis of ergosterol from isotopic acetate.

Abstract In a previously coenzyme A-deficient yeast, which was subsequently enriched, 74–84% of the carbon atoms of ergosterol were derived from acetate. Using these yeast cells and labeled acetate as the precursor, ergosterol of high specific activity can be prepared.