Donnan potentials from striated muscle liquid crystals. Sarcomere length dependence.

Donnan potentials from A-bands and I-bands were measured as a function of sarcomere length in skinned long-tonic muscle fibers of the crayfish. These measurements were made using standard electrophysiological technique. Simultaneously, the relative cross-sectional area of the fibers was determined. Lattice plane spacings and hence unit-cell volumes were determined by low-angle x-ray diffraction. At a sarcomere length at which the myosin filaments and actin filaments nominally do not overlap, measurements of potential, relative cross-sectional area, and unit-cell volume were used in conjunction with Donnan equilibrium theory to calculate the effective linear charge densities along the myosin filament (6.6 X 10(4) e-/mu) and actin filament (6.8 X 10(3) e-/mu). Using these linear charge densities, unit-cell volumes and Donnan equilibrium theory, an algorithm was developed to predict A-band and I-band potentials at any sarcomere length. Over the range of sarcomere lengths investigated, the predicted values coincide with the experimental data. The ability of the model to predict the data demonstrates the applicability of Donnan equilibrium theory to measurements of electrochemical potential from liquid-crystalline systems.     

Purification of d'Anjou Pear (Pyrus communis L.) Polyphenol Oxidase

Polyphenol oxidase (PPO) was extensively purified to homogeneity from d'Anjou pear (Pyrus communis L.) by extraction in the presence of the phenolic binder AG 2-X8 andTriton X-100. Chlorophyll pigment was removed by chromatography resulting in a clear, colorless enzyme extract. Purification of pear PPO was achieved after chromatography on Phenyl Sepharose CL-4B, DEAE-cellulose, and hydroxylapatite columns. Only after the columns were run at room temperature rather than at 4°C were sharp peaks and good resolution obtained. Reproducibility of the entire scheme was excellent with chromatography on the hydrophobic resin as a key to successful purification. Three separate fractions of pear PPO were homogeneous when stained for protein with the silver stain after polyacrylamide slab gel electrophoresis and corresponded to relative mobilities of 0.41, 0.43, and 0.73. The effect of dimethylsulfoxide on enzyme activity was investigated and found to increase significantly the activity of purified pear PPO over the control.     

An interaction between season of calving and nutrition on the resumption of ovarian cycles in post-partum beef cattle

Angus cows were first mated at approximately 27 months of age in 2 herds, calving 21 July to 15 September (Group E) or 9 September to 30 October (Group L). The cows were fed a high (H) or medium (M) plane of nutrition for 55 days before and 40 days after calving. There was a mean liveweight difference of 35 kg between cows in Groups EH + LH and Groups EM + LM immediately after calving and at 40 days after calving. Immediately after calving cows in Groups EH + EM were 11 kg heavier than cows in Groups LH + LM, but there was no difference at 40 days after calving. There was a significant interaction between calving time and nutrition in the return of cyclic ovarian function assessed from both interval to first oestrus and first elevated progesterone concentration. Mean intervals from calving to first oestrus were 66.7, 82.7, 56.7 and 62.3 days in Groups EH, EM, LH and LM respectively. These data demonstrate that season of calving influences resumption of ovarian cycles even at a constant high plane of nutrition and that season of calving interacts with nutrition such that effects of season are more likely to be expressed under conditions of low nutrition.     

Preliminary characterization of a Chinese hamster ovary cell glycosylation mutant isolated by screening for low intracellular lysosomal enzyme activity

A novel screening procedure was developed for isolating Chinese hamster ovary cell mutants altered in the early steps of the biosynthesis of asparagine-linked glycoproteins. This procedure identifies cells with low intracellular levels of two lysosomal hydrolases, beta-glucuronidase and alpha-iduronidase. One mutant cell line isolated in this way, CHB 11-1-3, has low intracellular levels of seven lysosomal enzymes as compared to wild-type cells. Although CHB 11-1-3 synthesizes mannosylphosphoryldolichol and [Man]₅[NAcG1cNH₂]₂-P-P-lipid, it fails to utilize these lipid intermediates to make normal amounts of [Glc]₃[Man]₉[NAcG1cNH₂]₂P-P-lipid. As a consequence of this glycosylation defect, this mutant transfers oligosaccharides of a different structure than wild type to the lysosomal enzyme beta-hexosaminidase. In addition, it underglycosylates its proteins.     

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.     

Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets

A murine monoclonal antibody, designated AP-2, reacts specifically with the complex formed by human platelet membrane glycoproteins IIb and IIIa, but does not react at all with the individual glycoproteins. Purified AP-2 covalently coupled to Sepharose CL4B was used as an immunoadsorbent column to purify the IIb-IIIa complex from a preparation of Triton X-100-solubilized human platelet proteins. Radioiodinated AP-2 was shown to bind to a single class of sites, with 57,400 +/- 9,700 molecules bound per cell (mean +/- S.D.) at saturation and a dissociation constant (Kd) of 0.64 +/- 0.15 nM (mean +/- S.D.). Binding could not be readily reversed even after a 1-h incubation with a 100-fold excess of cold antibody. AP-2 inhibits ADP-induced binding of radiolabeled fibrinogen to gel-filtered platelets in a noncompetitive fashion, consistent with the previous observation that AP-2 also inhibits the aggregation of platelets in plasma induced by a number of physiologic agonists, including adenosine diphosphate, epinephrine, collagen, thrombin, and arachidonic acid. Using AP-2, we have obtained evidence that the IIb-IIIa complex exists in the membrane of intact nonstimulated platelets and that complex integrity is not affected by external calcium ion concentration.     

Transposable Elements in Mendelian Populations. II. Distribution of Three COPIA-like Elements in a Natural Population of DROSOPHILA MELANOGASTER

Twenty X chromosomes isolated from a natural population of Drosophila melanogaster were surveyed using in situ hybridization to determine the number and cytogenetic location of three families of transposable elements: copia, 412 and 297. We found no sites of insertions in high frequency; in fact, frequencies of specific sites for all three elements were so low that each insertion could be interpreted as being unique. This suggests that rates of transposition and deletion for these elements are very high. Our data also show a higher than expected rate of the co-occurrence of different elements at the same site on the same chromosome.