The 'lysine cluster' in the N-terminal region of Na+/K(+)-ATPase alpha-subunit is not involved in ATPase activity.

The alpha-subunit of the Na+/K(+)-ATPases from several animal species have markedly similar amino acid sequences. However, the N-terminal sequences of the alpha-subunit are rather divergent except for lysine-rich sequences, the 'lysine cluster'. Here we report that the alpha-subunit from frog (Rana catesbeiana) has an N-terminal sequence with the 29 amino acid residues shorter than that of the Xenopus alpha-subunit deduced from its cDNA and hence lacks the 'lysine cluster'. Nevertheless, the Rana enzyme still exhibits ATPase activity. The ATP-dependent Na+ transport activity of the Rana enzyme was similar to that of the dog enzyme, which contains the 'lysine cluster'. Moreover, the Torpedo alpha-subunits deprived of the 'lysine cluster' by means of two gene deletions showed the same Na+/K(+)-ATPase activities as that of the wild type when expressed in Xenopus oocytes from their mRNAs. These results strongly suggest that the 'lysine cluster' in the N-terminal region of the alpha-subunit is not involved in the ATPase and ion transport activities. Since an active alpha-subunit was translated in Xenopus oocytes from mRNA lacking the N-terminal region including the 'lysine cluster', these regions were proved not to function as a membrane insertion signal sequence.     

Staurosporine: An Effective Inhibitor for Ca2+/Calmodulin-Dependent Protein Kinase II

We investigated the effect of staurosporine on Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10-100 nM) inhibited the activity of CaM kinase II. The half-maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (beta-casein). (c) Staurosporine suppressed the auto-phosphorylation of alpha- and beta-subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+/calmodulin-independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP-binding site or substrate-binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.     

Direct injection of hepatitis B virus DNA into liver induced hepatitis in adult rats

Hepatitis B virus (HBV) surface antigen (HBsAg) genes were injected directly into the liver of adult rats with non-histone chromosomal protein high mobility group 1 by the hemagglutinating B virus of Japan (Sendai virus)-liposome method (Kato, K., Nakanishi, M., Kaneda, Y., Uchida, T., and Okada, Y. (1991) J. Biol. Chem. 266, 3361-3364). Immunohistochemical analysis showed that HBV surface antigen was expressed by the hepatocytes in vivo. On successive injections of the HBsAg genes, the antibody to HBV surface polypeptides was produced in the rats, and characteristic pathological changes of lymphocytic focal necrosis and denaturation of hepatic cells were observed in the liver of all the rats. We conclude that hepatitis is caused by the direct injection of HBsAg genes.     

B cell response pathways regulated by IL-5 and IL-2. Secretory microH chain-mRNA and J chain mRNA expression are separately controlled events

We have examined the effect of IL-5 and/or IL-2 on the expression of the secretory form of microH chain (microsecond) and J chain mRNA in a homogeneous neoplastic B cell clone, in which proliferation, IL-2R up-regulation and entry into the IgM secretory state are separately controlled events. The IL-5 signal triggers a partial induction of CL-3 cells into the IgM secretory state, characterized by a striking increase of microsecond mRNA expression and an increase in the ratio of the secretory to membrane forms of microH chain mRNA, with a modest increase of J chain mRNA. In contrast, amplification of J chain mRNA is accomplished by the late-acting B cell differentiation stimulus, IL-2, acting on IL-5-pretreated CL-3 cells or acting simultaneously with IL-5 on CL-3 cells. Such dually stimulated cells now are fully induced into IgM secreting cells. These results define the relative roles of IL-5 and IL-2 in B cell differentiation by showing important regulatory effects at the mRNA level. In addition, these results substantiate that appearance of mRNA for J chain, a molecule key to the formation of pentameric IgM, is a limiting factor for high level IgM secretion. The separate control of microsecond and J chain mRNA found in CL-3 cells stimulated with IL-5 and IL-2 elucidates a molecular mechanism by which these two lymphokines synergize in the development of CL-3 cells into IgM secreting cells.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.     

Aminopeptidase M from human liver. II. Kinetic analysis of inhibition of the enzyme by bile acids

The mechanism of inhibition of aminopeptidase M by bile acids was analyzed by application of the specific velocity plot that was introduced by Baici [Eur. J. Biochem. 119, 9-14 (1981)]. Kinetic studies with three bile acids (cholic acid, deoxycholic acid, and chenodeoxycholic acid) and three substrates (Leu-Met, Leu-Gly, and Leu-pNA) showed that the inhibition constants Ki for the bile acids were appreciably different from each other, but that the Ki for each was not affected by the substrates used, being 0.89-1.03 mM for cholic acid, 0.42-0.66 mM for deoxycholic acid, and 0.24-0.31 mM for chenodeoxycholic acid. The values of the kinetic coefficient alpha [(apparent Ks in the presence of inhibitor)/Ks] for cholic acid with Leu-Met and Leu-Gly were 9.0 and 2.5, respectively. These values were very similar to those for chenodeoxycholic acid (7.0 and 2.7) but smaller than those for deoxycholic acid (21 and 11). The values of the other kinetic coefficient beta [(apparent kp in the presence of inhibitor)/kp] were 0 except in the case of the combinations of Leu-Gly with cholic acid (0.33) and Leu-Gly with chenodeoxycholic acid (0.13). On the basis of these kinetic parameters, the inhibitions by bile acids were classified into 4 types: competitive-noncompetitive linear mixed type (1 less than alpha less than infinity, beta = 0), noncompetitive-uncompetitive linear mixed type (0 less than alpha less than 1, beta = 0), pure noncompetitive type (alpha = 1, beta = 0), and hyperbolic mixed type (1 less than alpha less than infinity, 0 less than beta less than 1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification, microheterogeneity, and stability of human lipid transfer protein

A method for the purification of lipid transfer protein (LTP) from human plasma was developed with the aid of succinylated low density lipoprotein-Sepharose affinity column chromatography. The purified LTP exhibited a single main band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, upon isoelectric focusing on polyacrylamide gel, the preparations consistently showed nine bands with isoelectric points ranging from 4.6 to 5.4. The treatment of LTP with Clostridium perfringens neuraminidase shifted these multiple bands toward higher pH regions due to the release of sialic acid. Extensive treatment with neuraminidase resulted in the appearance of a major band with the isoelectric point of 5.6. The purified LTP was rapidly inactivated upon incubation at 37 degrees C due to the denaturation at the "air"-water interface. Various factors promoting or preventing this interfacial denaturation were elucidated. When purified LTP was stored at 4 degrees C, plasma neuraminidase co-purified with LTP became activated, resulting in the gradual desialylation of LTP. It seemed that the LTP preparations of apparent homogeneity are associated with a trace amount of an inactive form of plasma neuraminidase. The inclusion of 4 mM 2-mercaptoethanol or 0.2% EDTA in the storage media completely prevented the activation of plasma neuraminidase. These agents, however, did not significantly inhibit the already activated neuraminidase. When LTP was stored at -20 degrees C in very low ionic strength media, such as 0.001% EDTA (pH 7.4) and at high protein concentrations, the loss of the activity was minimal even after prolonged storage.     

Chromosomal location of the gene encoding the murine acute-phase protein serum amyloid P-component (SAP)

A full-length cDNA clone, pmSAP3, encoding the serum P component (SAP), has been used to search for DNA fragment length variation among mouse strains previously analyzed for differences in endogenous SAP levels. Three alleles were found usingEcoRI-digested DNA. The finding of a single 5.4-kb fragment, alleled, in DNA from DBA/2J mice suggests the presence of a singleSap locus. Segregation of DNA fragment associated withSap ^b andSap ^d alleles was analyzed in three sets of recombinant inbred (RI) strains. The strain distribution pattern found for theSap alleles was identical to that of alleles ofLy-9 in 43 individual RI strains, suggesting tight linkage withLy-9 on mouse chromosome 1. In the BXD RI strains, the SDP of theSap locus, defined by the difference in the endogenous SAP level, is also identical to the SDP of the DNA fragments. We propose to redesignate theSap locus to include both the structural element defined by the DNA polymorphism and the regulatory element involved in the regulation of SAP synthesis. TheSap locus is the major genetic element contributing to the regulation of SAP production. Other genetic factors are also involved, as shown by the presence of nonparental phenotypes in the individual BXH RI strains. This study was performed through special Coordination Funds of the Science and Technology Agency of the Japanese Government and PHS Grant GM24464 to R.W.E.     

Hypericin and pseudohypericin specifically inhibit protein kinase C: possible relation to their antiretroviral activity

Hypericin and pseudohypericin which have been isolated from plants of the Hypericum family are aromatic polycyclic diones. Daniel Meruelo et. al. have reported that hypericin and pseudohypericin showed potent antiretroviral activity including anti-human immunodeficiency virus (1,2). However, the mechanism of these antiretroviral activities has not been clarified. In the course of screening specific inhibitors of protein kinase C we have found that both compounds specifically inhibit protein kinase C with IC50 values 1.7 micrograms/ml and 15 micrograms/ml, respectively, and show antiproliferative activity against mammalian cells. These data suggest that antiretroviral activity of hypericin and pseudohypericin could be attributable to the inhibition of some phosphorylation involved by protein kinase C during viral infection of cells.     

Ab initio molecular orbital study on the effects of zinc ion,its ligands and ionic amino acid residues for proton transfer energetics between glu 270 and Zn co-ordinated water molecule in carboxypeptidase A

Thezinc-water-Glu 270 system was reported from the X-ray crystallographic study of native carboxypeptidase A(CPA) (Lipscomb et al., 1968). General base catalysis by the γ-carboxylate of Glu 270 was proposed for peptidase activity of CPA. The effects of zinc ion and its ligands (Glu 72, His 69-Asp 142, His 196) for proton transfer between Glu 270 and Zn co-ordinated water molecule in CPA were studied by the ab initio SCFLCAO-MO method. The results show that the proton transfer from the Zn co-ordinated water molecule to the γ-carboxylate of Glu 270 is greatly promoted by the Zn ion and, conversely, is greatly inhibited by its ligands. The facilitation effect of Zn ion and the inhibition effect of its ligands for the proton transfer were analysed by using the energy decomposition analysis. Moreover, calculations including all side chains of ionic amino acid residues and main chain residues in CPA as point fractional charges were performed. The results show that the proton transfer is affected by the ionic amino acid residues and is not affected by the main chain residues.