High affinity, calcium-stimulated adenosine triphosphatase activity in the particulate fraction of rat pancreatic acini

Adenosine triphosphatase activity which is Mg2+-dependent and stimulated by submicromolar concentrations of Ca2+ (as Ca . ATP) was identified in the total particulate fraction of rat pancreatic acini. Half-maximal activity (V0.5) is obtained at 100.1 +/- 6 nM Ca . ATP with a Hill coefficient of 2.2 +/- 0.1 (mean +/- S.E.; n = 4). Maximal activity was 75 +/- 19 pmol of Pi released from ATP minute-1 microgram of membrane protein-1 (mean +/- S.E.; n = 7). High affinity Ca2+-ATPase activity was unaffected by ouabain, Na+, K+, La3+, and added calmodulin. Activity was slightly reduced by ruthenium red (0.1 mM) and by oligomycin (80 micrograms/ml) but was reduced almost 50% by the phenothiazine derivative fluphenazine in a dose-related and Ca2+-dependent manner. Hydrolysis of p-nitrophenyl phosphate was 9% of the rate of ATP hydrolysis and was independent of Ca2+ concentration. However, ADP, GTP, UTP, and ITP were hydrolyzed at 76-93% the rate that ATP was hydrolyzed with V0.5 values and Hill coefficients similar to those of Ca . ATP. We conclude that rat pancreatic acini contain an enzyme for active Ca2+ translocation: ATPase activity that is Mg2+-dependent and stimulated by submicromolar concentrations of Ca . ATP. Substrate hydrolysis appears to involve positive cooperative interactions of multiple ligand-binding sites and may be regulated in part by calmodulin.     

Use of dextran and post-primary antibody fixation in immunoperoxidase staining of fresh frozen tissue. Detection of immunoglobulin associated with squamous carcinomas of the head and neck

Use of unfixed fresh frozen tissue sections for immunocytochemical studies reduces the possibility of denaturation of antigenic determinants compared to formalin fixation and paraffin embedding procedures. However, tissue and cellular morphology can be extensively altered in the numerous application and washing steps with frozen tissue sections. We tested a number of buffer solutions and showed that the use of dextran-containing buffers and fixation by glutaraldehyde after primary antibody application preserves tissue morphology. The procedures described here are also applicable to ascertaining the presence of Fc receptors of leukocytes in sections of carcinoma tissues. The buffered dextran washes and post-primary antibody fixation method was used to demonstrate the presence of immunoglobulin associated with squamous carcinoma cells. The immunoglobulin was not removed by washing of tissue sections at 37 degrees C but could be removed by low or high pH buffer washes, suggesting that the immunoglobulin is bound in a specific manner.     

Studies on ultrastructure and host range of aChlorella attacking virus

Summary A tail-less polygonal virus with a prominent capsid of about 140–150 nm in diameter and about 14–15 nm in thickness has been isolated from a freshwater pond. It shows a marked host specificity in attacking only an endosymbioticChlorella sp. isolated fromParamecium bursaria (Ciliata). Viral replication starts in the algal cytoplasm and both autospores and old cells are lysed. The ecology of the virus in the freshwater habitat is discussed. Screening tests for further phycoviruses were not successful.     

Structure-activity relationships of the yeast alpha-factor

The yeast Saccharomyces cerevisiae produces a peptide pheromone, termed the alpha-factor, as a prelude to sexual conjugation. Haploid MAT alpha-cells, but not haploid MAT a-cells or MAT a/alpha-diploids, produce this tridecapeptide of the structure: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr. Structural analogues of the alpha-factor have been prepared with alterations in many of the residues, derivatized peptides have been synthesized, and truncated and elongated peptides have been studied. These peptides have been analyzed for their biological activities by various assays. Mutants of S. cerevisiae have been isolated that do not respond to alpha-factor or are supersensitive to the pheromone and its analogues. The mating system of S. cerevisiae provides a powerful model in which genetics, biochemistry, and molecular biology can be used to unravel the mysteries of peptide hormone structure and function.     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Neutral and Acid Sphingomyelinases of Rat Brain: Somatotopographical Distribution and Activity Following Experimental Manipulation of the Dopaminergic System In Vivo

The activities of neutral, magnesium-stimulated, and acid sphingomyelinases were measured in five regions of rat brain. Neutral enzyme activity was 2-3-fold higher in striatum than in parietal cortex and 13-fold higher than in cerebral white matter. Acid sphingomyelinase activity was more evenly distributed throughout these regions. Striatal neutral sphingomyelinase activity was not affected by treatment of rats with reserpine or haloperidol and was reduced (16%) by 6-hydroxydopamine. Striatal acid sphingomyelinase was unaffected by reserpine and 6-hydroxydopamine, and was increased (17%) by haloperidol. We conclude that neutral, magnesium-stimulated sphingomyelinase activity differs in various regions of rat brain and is particularly enriched in the corpus striatum. However, it appears to be a constitutive component of tissue rather than a readily modulated regulatory element of the catecholaminergic system.     

Purification and characterization of hydroxypyruvate reductase from cucumber cotyledons

Hydroxypyruvate reductase (HPR), a marker enzyme of peroxisomes, has been purified to homogeneity from cotyledons of light-grown cucumber seedlings (Cucumis sativus var. Improved Long Green). In addition, the peroxisomal location of both HPR and serine-glyoxylate aminotransferase has been confirmed in cucumber cotyledons. The isolation procedure involved Polymin-P precipitation, a two-step precipitation with ammonium sulfate (35 and 50% saturation), affinity chromatography on Cibacron Blueagarose, and ion-exchange chromatography on DEAE-cellulose. HPR was purified 541-fold to a final specific activity of 525 ± 19 micromoles per minute per milligram of protein. Enzyme homogeneity was established by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was 91 to 95 kilodaltons, approximately double the apparent subunit molecular weight of 40,500 ± 1,400. With hydroxypyruvate as substrate, the pH optimum was 7.1 and K_m values were 62 ± 6 and 5.8 ± 0.7 micromolar for hydroxypyruvate and NADH, respectively. With glyoxylate as substrate, the pH optimum was 6.0, and the K_m values for glyoxylate and NADH were 5700 ± 600 and 2.9 ± 0.5 micromolar, respectively. Antibodies to HPR were raised in mice (by the ascites tumor method) and in rabbits, and their monospecificity was demonstrated by a modified Western blot immunodetection technique.     

The effect of reversal of Na and K electrochemical potential gradients on the active transport of amino acids in Ehrlich ascites tumor cells

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1. The net uptake of α-aminoisobutyric acid (AIB) in Ehrlich ascites tumor cells has been studied under a variety of transmembrane concentration gradients of Na⁺, K⁺ and AIB itself.     

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.