A hyperthermostable pullulanase produced by an extreme thermophile,Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability

Summary A cell-associated pullalanase (-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55 000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr-Ala-Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75–80° C and at 80–85° C, respectively. The enzyme was stable up to 90° C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae B. acidopullulyticus B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.Presented in part at the Annual Meeting of the Agricultural Chemical Society of Japan at Tokyo, April 2, 1987 (Abstracts, p 91)Offprint requests to: Y. Suzuki     

Conformational dynamics of DNA affected by intercalation and minor groove binding: direct observation of large DNA.

Using fluorescence microscopy, we have observed moving DNA molecules in solution and analyzed the "higher-order" structure in a quantitative manner. It was found that EB (ethidium bromide), an intercalator, has the effect to increase the persistent length. In other words, EB expands DNA. Whereas, DAPI (4',6-diamidino-2-phenylindole), a minor groove binding drug, decreases the persistent length. It is demonstrated that the direct observation of DNA molecules with fluorescence microscopy is quite useful to study the interaction of various chemical compounds with DNA molecules.     

Repair of a synthetic abasic site involves concerted reactions of DNA synthesis followed by excision and ligation.

A synthetic analog of an abasic site in DNA is efficiently repaired by a short-patch repair mechanism in soluble extracts of Xenopus laevis oocytes (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989). We present a detailed analysis of the repair mechanism, using extracts depleted of endogenous nucleotide pools. ATP was required for repair with a sharp optimal concentration of 5 mM. The initial rate of repair was increased by preincubation of the DNA in the extract in the presence of ATP. During this preincubation, the DNA was cleaved on the 5' side of the lesion by a class II apurinic-apyrimidinic endonuclease, but removal of the abasic sugar residue was not observed prior to addition of deoxynucleotides to the reaction. Immediately following DNA synthesis, excision and ligation proceeded in a coordinated manner to complete repair. DNA preincubated in the extract in the absence of deoxynucleotides remained associated with repair enzymes during gel filtration. These observations suggest that the enzymes involved in concerted repair of the abasic site form a complex on DNA.     

Altered localization of antigen recognized by proteinuriainducing monoclonal antibody in experimental nephrosis

Change in the localization of the antigen recognized by the proteinuria-inducing monoclonal antibody (MA) 5-1-6 in experimental nephrosis was studied by indirect and biotin-avidin immunofluorescence, and immunoperoxidase at light and electron microscopical levels. The proteinuric state was induced by the administration of the aminonucleoside of puromycin (PAN) or adriamycin. The antigen decreased in quantity and/or its distribution changed with an increase in the amount of protein excreted in both experimental models. Recovery from the alterations observed during the development and proteinuria appeared to occur when PAN-induced proteinuria subsided. This antigenic molecule may thus be essential for maintaining the normal permselectivity of glomerular capillary walls.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region.

A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.     

Comparative anatomical study of arteriographs of the hand of primates. Deep palmar arterial arches and their correlating arteries in cercopithecidae, pongidae and hominidae

A three-dimensional angiographic analysis of the deep palmar arterial arches and their correlating arteries in Cercopithecidae, Pongidae and Hominidae revealed the following features. In Cercopithecidae, 3 deep palmar arches are formed by the perforating branches of the 2nd dorsal metacarpal artery: 2 proximal arches (the catella volaris proximalis and the arcus volaris profundus) and 1 distal arch (the catella volaris distalis). The intermetacarpal arteries arise from the catella volaris proximalis and the palmar metacarpal arteries arise from the arcus volaris profundus. In Pongidae, the arches are formed by the perforating branches of the 1st dorsal metacarpal artery, and they are composed of only the catella volaris proximalis and catella volaris distalis. In Hominidae, the arches are formed by the perforating branches of the 1st dorsal metacarpal artery, and they consist of the arcus volaris profundus and an incomplete catella volaris distalis.     

Expression of sodium pump activities in BALB/c 3T3 cells transfected with cDNA encoding alpha 3-subunits of rat brain Na+,K+-ATPase

The cDNAs encoding alpha 3-subunits of rat brain Na+,K+-ATPase and the neomycin resistance gene were incorporated into BALB/c 3T3 cells by the co-transfection method. Stably transformed cells were selected with 300 micrograms/ml of neomycin (G-418) for 6 weeks. Northern blot analysis using the 3'-non-translated region of the cDNA as a probe revealed that the alpha 3 mRNA appeared in transfected cells. Na+,K+-ATPase activity of the transfected cells was twice that of wild-type cells. Regarding ouabain sensitivity, the Na+,K+-ATPase showed two Ki values for ouabain (8 x 10(-8) and 4.5 x 10(-5) M) in transfected cells while wild-type cells displayed only the higher value. Ouabain sensitivity of Rb+ uptake also demonstrated two Ki values in the transfected cells (8 x 10(-8) and 4 x 10(-5) M) and a Ki in wild-type cells of 4 x 10(-5) M. It is concluded that alpha 3 is a highly ouabain-sensitive catalytic subunit of Na+,K+-ATPase. It is also suggested that ouabain sensitivity is exclusively determined by the properties of the alpha-subunit rather than the beta-subunit. This is the first report on the catalytic characteristics of the alpha 3 isoform of Na+,K+-ATPase.