An electrogenic proton pump associated with the Golgi apparatus of mouse liver driven by NADH and ATP

Golgi-apparatus membranes, isolated from mouse liver, pump protons inwards, when supplied with NADH or ATP. The acidification of Golgi-apparatus cisternae and vesicles was detected with neutral red, a permeant dye, as a difference in absorbance at 550 nm minus that at 600 nm. The maximum rates detected with NADH and ATP were between 0.0006-0.0009 and 0.0030-0.0050 delta OD units/mg of protein/min, respectively, at pH 7.5. The outside buffer used was a bovine serum albumin suspension. The acidification of Golgi apparatus was inhibited from 45 to 100% by ionophores and from 22 to 100% by uncouplers. The results implicate both ATP and a redox system coupled to NADH oxidation in the acidification of Golgi-apparatus membranes.     

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk^– cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.     

A Calcium-Selective Site in Photosystem II of Spinach Chloroplasts

After acid-treatment of spinach (Spinacia oleracea) chloroplasts, various partial electron transport reactions are inactivated from 25 to 75%. Divalent cations in concentrations from 10 to 50 millimolar can partially restore electron transport rates. Two cation-specific sites have been found in photosystem II: one on the 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-insensitive silicomolybdate pathway, which responds better to restoration by Mg²⁺ than by Ca²⁺ ions, the other on the forward pathway to photosystem I, located on the 2,5-dimethylbenzoquinone pathway. This site is selectively restored by Ca²⁺ ions. When protonated chloroplasts are treated with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aziridine, a carboxyl group modifying reagent, presumed to react with glutamic and aspartic acid residues of proteins, restoration of electron transport at the Ca²⁺-selective site on the 2,5-dimethylbenzoquinone pathway is impaired, while no difference in restoration is seen at the Mg²⁺ site on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate pathway.

Trypsin treatment of chloroplasts modifies the light-harvesting pigment-protein complex, destroys the dibromothymoquinone-insensitive 2,5-dimethyl-benzoquinone reduction, but does not interfere with the partial restoration of activity of this pathway by Ca²⁺ ions, implying that the selective Ca²⁺ effect on photosystem II (selective Ca²⁺ site) is different from its effects as a divalent cation on the light-harvesting pigment-protein complex involved in the excitation energy distribution between the two photosystems.

     

Dielectrophoresis of Cells

Dielectrophoresis, the motion produced by the action of nonuniform electric field upon a neutral object, is shown to be a simple and useful technique for the study of cellular organisms. In the present study of yeast (Saccharomyces cerevisiae) using a simple pin-pin electrode system of platinum and high-frequency alternating fields, one observes that the collectability of cells at the electrode tip, i.e. at the region of highest field strength, depends upon physical parameters such as field strength, field uniformity, frequency, cell concentration, suspension conductivity, and time of collection. The yield of cells collected is also observed to depend upon biological factors such as colony age, thermal treatment of the cells, and chemical poisons, but not upon irradiation with ultraviolet light. Several interesting side effect phenomena coincident with nonuniform electric field conditions were observed, including stirring (related to “jet” effects at localized electrode sites), discontinuous repulsions, and cellular rotation which was found to be frequency dependent.     

A Lipid Requirement for Photosystem I Activity in Heptane-extracted Spinach Chloroplasts

A lipid requirement for photosystem I activity in Spinacia oleracea chloroplasts has been characterized. The transfer of electrons from tetramethyl-p-phenylenediamine through the chloroplast photosystem to viologen dye was used as an assay of photosystem I activity. Activity is diminished by prolonged heptane extraction and is partially restored by readdition of the extracted lipid. Extracted chloroplasts require plastocyanin for maximal restoration of activity. The effect of lipid extract in restoration is partially replaced by triglycerides containing unsaturated, C₁₈ fatty acids. Various potential redox carriers which occur naturally in chloroplasts do not substitute for extracted lipid. Galacto-lipids, sulfolipids, and phospholipids are not involved in the restoration of activity.     

Biology of the small pelagic fishes in the New Volta Lake in Ghana. Part III: Sex and reproduction

Reproductive patterns of five species, from three families, of small pelagic fishes in the new Volta Lake in Ghana showed several similar features. These included sex size defferences, males maturing and reaching final sizes smaller than females; heterogenous sex ratios in four species, being most markedly unequal usually at peak breeding times; demersal eggs; and extended fractional spawning patterns. The clupeids Pellonula and Cynothrissa and the cyprinid Barilius all breed in the dry season, whereas the schilbeids Physailia and Siluranodon are wet-season spawners. Only Pellonula and Physailia have extended their breeding season in the new lake to cover most of the year; the other species have not, although Siluranodon spawns into the early dry season. In the lake environment Pellonula's pelagic larva is no longer endangered by seasonal floods, whereas Physailia can use the lake's continuous “high waters” for extended breeding. Relaxation of breeding patterns and protracted fractional spawning has allowed best exploitation of the new regime.     

Activation in a Skeletal Muscle Contraction Model with a Modification for Insect Fibrillar Muscle

A sliding filament model for muscle contraction is extended by including an activation mechanism based on the hypothesis that the binding of calcium by a regulating protein in the myofibrils must occur before the rate constant governing the making of interactions between cross-bridges and thin filament sites can take on nonzero values. The magnitude of the rate constant is proportional to the amount of bound calcium. The model's isometric twitch and rise of force in an isometric tetanus are similar to the curves produced by real muscles. It redevelops force after a quick release in an isometric tetanus faster than the initial rise. Quick release experiments on the model during an isometric twitch show that the “active state” curve produced is different from the postulated calcium binding curve. The force developed by the model can be increased by a small quick stretch delivered soon after activation to values near the maximum generated in an isometric tetanus. Following the quick stretch, the force remains near the tetanic maximum for a long time even though the calcium binding curve rises to a peak and subsequently decays by about 50%. The model satisfies the constraint of shortening with a constant velocity under a constant load. Modifications can be made in the model so that it produces the delayed force changes following step length changes characteristic of insect fibrillar muscle.     

Assaying Actual Hematein Content of Commercial Hematoxylins and Hemateins

Hematein-free hematoxylin (HFH) was prepared by a modification of the procedure of Palmer and Lillie (Histochemie, 5: 44-54, 1965). Fifty mg of HFH were dissolved in 5 mg of ethylene glycol and then 45 nil of an aqueous solution of 2.25 gm KAl(SO₄)₂. 12H₂O and 5.445 mg KIO₃ were added. Since this amount of KIO₃ would be sufficient to oxidize 25 mg of HFH to hematein we have termed this half-oxidized hematoxylin (HOH). The peak absorbance (560 nm) of this purple solution remained constant for at least a week. With omission of the KIO₃ the solution was colorless. A curve was constructed by plotting absorbance against concentration of hematein in HOH at various dilutions. For analyses of hematein content of commercial hematoxylins 50 mg of sample and 100 mg of hydroquinone were dissolved in 5 ml of ethylene glycol and then 45 ml of a 5% solution of KAl(SO₄)₂. 12H₂O were added. The addition of the hydroquinone stabilized the absorbance for about 5 min. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. Eleven samples of hematoxylin certified by the Biological Stain Commission had hematein concentrations varying from 0.01 to 0.43%. For analyses of the available hematein content of commercial hemateins, 50 mg of sample were dissolved in 10 ml of ethylene glycol, then 45 ml of water and 45 ml of 5% KAI(SO₄)₂. 12H₂O added. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. In 9 samples of hematein from 4 different sources the active hematein content varied from 19 to 97%.     

Multiple forms of porcine pancreatic α-amylase

Porcine pancreatic α-amylase can be fractionated into two components by DEAE-cellulose chromatography and by disc electrophoresis. The basis for fractionation is tentatively ascribed to a charge difference. The two components displayed the same specific activity and their thermal and pH stability, as well as the variation of V_max and K_m with pH, were identical within experimental error. It is concluded that the multiple forms of the amylase are physically distinct, but structurally related, with a common active site.