Enzyme systems involved in phosphorylation and dephosphorylation of two endogenous phosphoproteins in neuronal membranes.

Adenosine 3′:5′-monophosphate-dependent protein kinase and phosphoprotein phosphatases were solubilized by Triton X-100, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes, and partially purified. The properties of these partially purified enzymes were studied using two substrates, Protein I and Protein II, prepared from the synaptic membrane fraction, as well as the substrates protamine and histone. The results suggest that the phosphorylation of Protein I and Protein II, as well as protamine and histone, are catalyzed by a single species of cAMP-deperident protein kinase. Thus, a single peak of protein kinase activity was observed, upon DEAE-cellulose hromatography of the Triton X-100 extract of the synaptic membrane preparation, which catalyzed the phosphorylation of all four substrate proteins. Moreover, the activity of this partially purified protein kinase toward the various substrate proteins was altered in a parallel fashion, either when the protein kinase preparation was subjected to heat inactivation or pH inactivation, or when the enzyme was assayed in the presence of various concentrations of cyclic nucleotides or of a protein kinase modulator. The individual protein substrates acted as competitive inhibitors with respect to one another. Upon sucrose density gradient centrifugation, the protein kinase activity toward the various substrates sedimented as a single peak. Finally, the relative specific activities toward the various substrates did not change significantly during a 2000-fold purification of the enzyme. In contrast to these observations with protein kinase, two peaks of protein phosphatase activity, with markedly different specificities toward Protein I and Protein II, were found upon DEAE-cellulose and Bio-Gel P-200 column chromatography of the Triton X-100 extract of the synaptic membrane fractions. One peak catalyzed the dephosphorylation of Phosphoprotein I but not of Phosphoprotein II, whereas the other peak catalyzed the dephosphorylation of Phosphoprotein II but not of Phosphoprotein I. The dephosphorylation of Phosphoprotein I by Phosphoprotein I phosphatase was not affected by adenosine 3':5'-monophosphate, whereas the dephosphorylation of Phosphoprotein II by Phosphoprotein II phosphatase required the presence of this nucleotide. Moreover, the two phosphatases differed from one another with respect to Stokes' radius as well as sedimentation coefficient.     

A dominant interfering mutation (CYR3) of the Saccharomyces cerevisiae RAS2 gene.

The dominant cyclic AMP-requiring mutation CYR3 had been previously reported as a mutation in the regulatory subunit of cyclic AMP-dependent protein kinase. However, recharacterization revealed that the CYR3 mutation was a nonconditional dominant lethal mutation and was a missense allele of RAS2 which results from the substitution of aspartic acid for glycine at amino acid 22.     

INTERACTIONS BETWEEN KURIL SEALS AND SALMON TRAP NET FISHERY IN THE COASTAL WATERS OF SOUTHEASTERN HOKKAIDO

An annual average of 163 Kuril seals was found dead in two years in salmon trap nets along the coastal waters of the Nemuro Peninsula and adjacent areas. The seal-caused damage to the total salmon catch at the salmon trap nets was concentrated in some of them, particularly No. 27, where seals killed or injured 5.1% of the catch in 1982, and 1.8% in 1983. Based on the proportion of Kuril seals among the dead seals in the trap nets, it was estimated that Kuril seals damaged 4.7% of the total salmon catch at No. 27 in 1982, and 1.7% in 1983. Not all seals that entered the trap net drowned; some killed or damaged salmon, and then escaped.     

The functional domain of adenylate cyclase associated with entry into meiosis in Saccharomyces cerevisiae.

Diploid yeast cells that carry a part of the CYR1 gene deficient in a region coding for the N-terminal domain of adenylate cyclase were growth arrested and accumulated unbudded cells after inoculation into complete medium or nitrogen-free medium, but produced many cells which had one or more buds after incubation in sporulation medium. The cells incubated in sporulation medium had abnormal spindles which were free from the spindle pole bodies, larger in size, or frequently distributed in cytoplasm. The levels of cyclic AMP in these cells did not decrease to the wild-type level after transfer to the sporulation medium and remained at a constant level. The results suggest that the N-terminal domain of adenylate cyclase is associated with the regulatory function for sporulation. The environmental signals for sporulation may be transferred to the adenylate cyclase system through a factor that negatively interacts with the N-terminal domain of this enzyme.     

Characterization of rabbit liver P450IIE1 synthesized in transformed yeast cells

cDNA for chimeric protein, P450(3P4), consisting of the amino-terminal 43 residues (the membrane-anchor region) of rabbit P450IIC14 and the remaining 447 residues of rabbit P450IIE1 was constructed, then cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. P450(3P4) thus synthesized in the transformed yeast cells was partially purified, and its spectral and catalytic properties were examined. In the oxidized state P450(3P4) exhibited a high-spin type absorption spectrum even in the absence of a substrate. The reduced CO complex of the P450 showed a Soret absorption maximum at 452 nm. P450(3P4) catalyzed aniline p-hydroxylation, N-nitrosodimethylamine demethylation, benzphetamine N-demethylation, and laurate and caprate (omega-1)-hydroxylation in the reconstituted system containing the P450 and NADPH-P450 reductase. These results indicate that P450(3P4) preparation obtained from the transformed yeast cells has spectral and catalytic characteristics identical with those of P450IIE1 purified from rabbit liver microsomes, confirming the substrate specificity reported of P450IIE1.     

Characterization of cyclic AMP-requiring yeast mutants altered in the regulatory subunit of protein kinase

The CYR3 mutant of yeast, Saccharomyces cerevisiae, partially accumulated unbudded cells and required cAMP for the best growth at 35 degrees C. The CYR3 mutation was partially dominant over the wild type counterpart and suppressed by the bcy1 mutation which is responsible for the deficiency of the regulatory subunit of cAMP-dependent protein kinase. The molecular weights of cAMP-dependent protein kinase and its catalytic and regulatory subunits were 160,000, 30,000, and 50,000, respectively. No significant differences in the molecular weights of cAMP-dependent protein kinase and the subunits were found between the wild type and CYR3 mutant strains. However, the cAMP-dependent protein kinase activity of CYR3 cells showed significantly higher Ka values for activation by cAMP at 35 degrees C than those of wild type and a clear difference in the electrophoretic mobility of the regulatory subunit was found between the wild type and CYR3 enzymes. The CYR3 mutation was suppressed by the IAC mutation which caused the production of a significantly high level of cAMP. The results indicate that the CYR3 phenotype was produced by a structural mutation in the CYR3 gene coding for the regulatory subunit of cAMP-dependent protein kinase in yeast.     

Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites

The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca²⁺ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca²⁺. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.     

Conformational studies of poly-beta-1-naphthylmethyl-L-aspartate.

Poly-β-1-naphthylmethyl-L -aspartate and copolymers of β-1-naphthylmethyl-L -aspartate and γ-benzyl-L -glutamate were prepared. From the results obtained by a study of infrared and circular dichroism spectra, poly-β-1-naphthylmethyl-L -aspartate was found to be a left-handed α-helix both in the solid state and in solution. The fluorescence spectra of these polymers showed excimer emission of the naphthyl chromophores and gave some information about the arrangement of the side-chain chromophores. By optical titration experiments, it was found that an increasing amount of β-1-naphthylmethyl-L -aspartate residues in the copolymers induces a progressive instability of the helical structure.     

How is the flagellar length of mature sperm determined? I. Comparison of flagellar growth in spermatids between newt and Xenopus in vitro

The kinetics of flagellar growth in round spermatids were compared between Xenopus laevis and Cynops pyrrhogaster in vitro, the latter of which has about 13 times longer flagella in mature sperm than the former. In both species, more than 90% of the spermatids derived from marked primary spermatocytes grew flagella. In Xenopus the average flagellar length increased to 28 microns by the 6th day and then stopped growth, while in the newt, flagellar growth did not stop until reaching 107 microns in average on the 10th day. Maximal length was 36-38 microns in Xenopus and 187 microns in the newt. Two major differences in kinetics of flagellar growth were found between the two species. First, the initial rate of growth in the newt was about double the rate in Xenopus. Second, the period of flagellar growth in the newt (10 days) was also about double the period in Xenopus (5-6 days). Actinomycin D (10 micrograms/ml) had no inhibitory effect on flagellar growth in either species, whereas cycloheximide (10 microM) inhibited flagellar growth by more than 80% in both species. These results indicate that translational control presumably of flagellar protein synthesis plays an important role in flagellar growth in both species and in the difference in flagellar length in spermatids between Xenopus and newt.     

Cleome hassleriana plants fully support the development and reproduction of Nesidiocoris tenuis

BioControl - Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is a zoophytophagous predator that feeds on plants as well as prey. Several non-crop host plant species have been used to maintain...