Herbert Zipper

The exchange and maximal net flux of glucose across the human erythrocyte. II. The effect of two sulphydryl enzyme inhibitors, chlormerodrin and p-chloromercuribenzene sulfonic acid.

Abstract The exchange and maximal net fluxes of [14C]glucose across the membrane of the human red cell were measured. The effects of p- chloromercuribenzene sulfonic acid and chlormerodrin on these two parameters of glucose transport were determined. At low concentrations p- chloromercuribenzene sulfonic acid (a non-penetrating organic mercurial) was found to readily inhibit the exchange flux but not the net efflux. At extremely high concentrations of p- chloromercuribenzne sulfonic acid the maximal net efflux showed some degree of inhibition. Chlormerodrin (a penetrating organic mercurial) inhibited both the exchange and net fluxes in the same manner. The addition of insulin in certain instances reduced the degree of inhibition caused by the organic mercurials. Insulin had no effect on the amount of either p- chloromercuribenzene sulfonic acid or chlormerodrin which bound to the red cell. From the results obtained, it is suggested that there exist glucose-reactive sites on both the outer and inner surfaces of the membrane. The results also suggest a carrier system possessing different sites or molecular arrangements for glucose egrees and for glucose entry.

Cytochrome oxidase solubilization at high pH: I. Effects of pH, Ca2+ and Mg2+ on the reversible solubilization of cytochrome oxidase and cytochrome b by synthetic zeolites and by EDTA

Abstract Treatment of extensively water-washed beef heart or yeast mitochondria with synthetic A or X type zeolites in the Na + or K + form disaggregates the mitochondria and solubilizes cytochromes b , c , and oxidase in aqueous systems whose pH is between 10 and 11. However, when mitochondria are treated with the same types of A or X zeolites in the Ca 2+ or Mg 2+ forms, only solubilization of some cytochrome c occurs, and cytochromes b and oxidase remain insoluble in aqueous systems whose pH is between 8 and 9.5. Incubation of heart mitochondria with 0.05 m EDTA at pH 10.5 (or yeast mitochondria at pH values between 9 and 10) results in complexation of all of the mitochondrial Ca 2+ and Mg 2+ by the EDTA, and concomitantly, cytochromes b , c , and oxidase are solubilized. At lower pH values, the divalent cations are still complexed, but solubilization of oxidase and b components does not occur. Restoration of Ca 2+ (as chloride) to either zeolite- or EDTA-solubilized mitochondrial systems quantitatively precipitates the b , c , and oxidase components which can be centrifuged down at 2000 g for 10 min. However, restoration of Mg 2+ (as chloride) to either zeolite- or EDTA-solubilized mitochondrial systems causes slow formation of an aggregate which does not contain cytochrome b , c , or oxidase components, all of which remain in solution. Finally, when the high pH of zeolite- or EDTA-solubilized cytochrome b and oxidase systems is lowered by back-titration with acid, the above components are quantitatively precipitated.

Cytochrome oxidase solubilization at high pH: II. Separation and partial isolation of cytochrome oxidase from beef heart mitochondria without use of detergents or sonic oscillations☆

Abstract Two new methods are presented for the separation and partial isolation of cytochrome oxidase from high pH-solubilized beef heart mitochondria. In the first method, cytochromes c and b are extracted from extensively water-washed beef heart mitochondria by titration with alkali hydroxides to pH values between 10.0–10.2, leaving behind cytochrome oxidase-enriched residues. The oxidase-rich residues are washed with water and re-extracted by titration with alkali hydroxides to pH 12, yielding a soluble fraction rich in cytochrome oxidase and very low in content of cytochrome b and c components. The solubilized, oxidase-enriched preparations are treated with (NH 4 ) 2 SO 4 to obtain a cytochrome oxidase preparation which contains no cytochrome b and little or no cytochrome c . The material solubilized at pH 10.0–10.2 contains mainly b and c cytochrome components and only traces of the oxidase. The latter can be removed by pH adjustment to yield a fraction containing only b and c cytochromes and no a or oxidase components. In the second method, the extensively water-washed beef heart mitochondria are solubilized by alkali titration to pH 12. The solubilized material containing c , b , and oxidase components is spun at 110,0000 for 1 hr, yielding a residue containing cytochrome oxidase and very little or no b and c components. The above methods permit simple and direct physicochemical separations of cytochrome components from mitochondria and preparation of active oxidase components in good yield, without the use of bile salts, detergents, or sonic oscillations.

Development of chick limb bud chondrocytes in cell culture: morphologic and oxidative metabolic observations.

For the purpose of describing early chondrogenic metabolic and structural events, measurements were made of oxidative and other enzymatic activities at various stages in the morphologic development of chondrocytes over a period of 18 to 20 days in continuous cell culture. Comparisons were also made between cells grown in 20% O2 and in 35% O2. These cultures exhibited rapid confluence (within 24 hours), early development of cartilaginous nodules by Day 2 to 3 and metachromatic staining of the chondrocyte matrix by Day 3 to 4. Under 35% O2, cell sheets were thicker and there was increased pleomorphism of chondrocyte and fibroblast cell types, with a relative increase of fibroblast components and reduction in chondroblasts and chondrocyte aggregates. Using the von Kossa staining procedure, calcium salt deposition was observed by Day 9. There was no apparent difference in mineralization of cultures grown under the low and high O2 tensions. Under normoxic conditions cytochrome oxidase and malate dehydrogenase (MDH) activities increased rapidly for the first three to four days and then remained essentially constant. Lactate dehydrogenase (LDH) activity increased continuously over the life of the culture. Acid phosphatase increased rapidly until about Day 13 after which it remained constant, whereas alkaline phosphatase showed a bimodal activity profile. Under hyperoxic conditions, cytochrome oxidase, MDH and alkaline phosphatase activity were significantly inhibited. LDH and acid phosphatase activities were markedly inhibited initially but with time showed a degree of recovery.

Absence of mitochondrial terminal respiratory enzymes in cartilage matrix vesicles

SummaryThis study attempted to detect evidence of mitochondrial terminal respiratory components in matrix vesicles isolated from rachitic rat tibial epiphyseal plates. Biochemical assays for cytochrome c oxidase, NAD isocitrate dehydrogenase, NADP isocitrate dehydrogenase and succinate-cytochrome c reductase were negative. Polarimetric determinations revealed that the addition of succinate to matrix vesicles in suspension did not cause any increase in oxygen utilization. Spectrophotometric tracings of deoxycholate-solubilized matrix vesicles showed no characteristic absorption peaks or maxima belonging to any of the cytochrome complex components. Attempts to prepare pyridine hemochromes of cytochrome prosthetic groups from the matrix vesicles were also unsuccessful. The above results indicate that key components of mitochondrial respiratory systems are not detectable in rachitic matrix vesicles. The results are compatible with the interpretation that such vesicles are not derived from mitochondria.