Some Characteristics of Protein Kinases in Lemna paucicostata

The three protein kinases of Lemna paucicostata that are separableby DEAE-Sephacel chromatography have been designated PI, PIIand PIII [Kato et al. (1983) Plant & Cell Physiol. 24: 841].The optimum pH for the PI and PII enzymes was 7.5 and for thePHI enzyme 7.0. The activities of these enzymes were stimulatedby divalent cations, the maximum stimulation being producedby 5 nw Mg^{2 $} for PI, by 3 mM Co^{2 $} for PII and by 1 mM Mn^2$ for PIII. The cytokinins; benzyladenine, kinetin and zeatin,inhibited the activity of the PIII enzyme. The molecular weightsof the PI and PII enzymes did not change after incubation withcAMP even though their activities were regulated by this compound. (Received October 17, 1983; )     

Importance of methionine residues in the enzymatic carboxylation of biotin-containing peptides representing the local biotinyl site of E. coli acetyl-CoA carboxylase

A biotin-containing hexapeptide Ac-Glu-Ala-Met-Bct-Met-Met (1) that represents the local biotin-containing site of Escherichia coli acetyl-CoA carboxylase has been prepared by the solid phase method. Peptide 1 is carboxylated by the biotin carboxylase subunit dimer of E. coli acetyl-CoA carboxylase with the following kinetic parameters; Km 12 mM, Vmax 2.8 microM X min-1. These compare with the parameters for biotin of Km 214 mM and Vmax 28 microM X min -1. Hence, the overall reactivity (Vmax/Km) of 1 is 1.8 times greater than that of free biotin. When all methionines in 1 are replaced by alanine, the resulting peptide (2) retains a similar binding ability but with a much decreased Vmax. It was also found that peptide 3, which carries an N epsilon-benzyloxycarbonyllysine in place of biocytin in 1, decreases the Km of biotin threefold.     

Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.

Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.     

Constancy of rDNA Content during Dedifferentiation of Carrot and Jerusalem Artichoke Explants

When carrot explants were cultured with phytohormones, DNA synthesistook place synchronously in the explants and a satellite DNAwith a heavier density in CsCl than the bulk DNA replicatedin the earliest phase of the first replication period. The earlyreplicating carrot satellite consisted of a component havingan identical density to carrot rDNA and another component havinga density between the p-value of carrot rDNA and that of thebulk DNA. DNA-rRNA hybridization was used to explore the possibilitythat this early replication of the satellites leads to amplificationof rDNA in the explant cells, in which massive ribosome synthesisis known to occur. The results showed that there was neitheramplification nor underreplication of rRNA genes during callusformation and its growth. Experiments with explants of Jerusalem artichoke tuber, whichare well known as a synchronous replication system, showed thata component slightly heavier than the bulk DNA was synthesizedat the early phases of the first replication period. However,the density of this early replicating satellite differed fromthat of artichoke rDNA. DNA-rRNA hybridization experiments againshowed no gross changes of rDNA content during dedifferentiationof this plant system. (Received September 30, 1981; Accepted January 5, 1982)     

RNA synthesis in embryo axes of germinating pea seeds

RNA synthesis during germination was investigated by labelingpea embryo axes or seedling roots with radioactive uridine oradenosine. The results indicated that all RNA species of pre-rRNAs(ribosomal precursor RNAs), rRNAs, heterodisperse-type RNA and4–5S low molecular weight RNA were synthesized from the6th to 64th hour of the period examined. At the very early stageof germination, some conspicuous labeling of the heterodisperse-typeRNA was observed after pulse-labeling. There was no great differencein the labeling patterns of various RNA species with regardto other later stages. When embryo axes were labeled for 1 hrwith ³H-adenosine from the 16th hour, about 25% of the labeledwhole cell RNA was retained on the membrane filter. The ratioof labeled poly(A)-containing RNA, however, decreased as germinationproceeded. The poly (A)-containing RNA sedimented heterodisperselywith a mean value of about 20S in a sucrose density gradient;this size-distribution did not vary throughout germination. (Received January 16, 1979; )     

Selective assay of monomeric and filamentous actin in cell extracts,using inhibition of deoxyribonuclease I

A simple and selective assay for monomeric and filamentous actin is presented, based on the inhibition of DNAase I by actin. In mixtures of monomeric and filamentous actin, only the monomeric form is measured as DNAase inhibitor. The total amount of actin in a sample can be determined after depolymerization of F actin with guanidine hydrochloride. The assay is rapid enough to detect changes in the polymerization state of actin in vitro over time intervals as short as 3 min. Data characterizing unpolymerized and filamentous actin pools in extracts of human platelets, lymphocytes and HeLa cells are presented.     

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.