Molecular cloning of cDNA encoding the alpha unit of CNGC gene from human fetal heart

Cyclic nucleotide-gated ion channels (CNGCs) play crucial roles in visual and olfactory signal transduction. As a first step to explore the presence of a CNGC gene in human heart, we cloned a human heart CNGC gene. The sequence consists of 111 bp 5' non-coding region and a 2064 bp open reading frame which is followed by a 459 bp 3' non-coding region. The predicted protein consists of 688 amino acids with a short highly charged segment rich in lysine and glutamate. Sequence comparison indicates that the human heart cDNA is almost identical to the retinal rod photo receptor CNGC cDNA. However, the human cardiac cDNA is lacking a 205 bp Alu fragment in the 5'-uncoding region, has a glutamic acid residue at amino acid position 129, and has a replacement of glutamic acid with a lysine residue at amino acid position 99. Data obtained with northern blot analysis confirm the presence of RNA for the CNGC alpha chain. This channel might play a role in cyclic nucleotide-mediated cellular processes, such as the inotropic effect in the heart.     

Mutations affecting the alpha subunit of Bordetella pertussis RNA polymerase suppress growth inhibition conferred by short C-terminal deletions of the response regulator BvgA

The effects of short deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system were examined in Bordetella pertussis. When present as a single copy in the chromosome, deletions removing as few as two amino acids conferred a completely Bvg- phenotype. When provided in trans, on the broad-host-range plasmid pRK290, under the control of the native bvgAS promoter, deletions of two or three amino acids conferred a profound growth inhibition which was dependent on the integrity and activity of the wild-type chromosomal bvgAS locus. It is proposed that this phenotype was the result of an inappropriate interaction of the mutant BvgA protein with the RNA polymerase enzyme, specifically the alpha subunit. Mutant strains in which this growth inhibition was relieved were isolated and characterized. Although most of the suppressor mutations affected either the mutant plasmid copy or the wild-type chromosomal bvg locus, three mutations which affected the alpha subunit of B. pertussis RNA polymerase were also isolated. Two of these resulted in increased levels of the alpha subunit, and one caused a substitution of glycine for the aspartic acid residue at position 171, in the N-terminal domain. All three mutations also resulted in a differential phenotype in that expression of fha was essentially normal, but expression of ptx was greatly reduced.     

Site-directed mutagenesis of the yeast V-ATPase A subunit

To investigate the function of residues at the catalytic nucleotide binding site of the V-ATPase, we have carried out site-directed mutagenesis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast. Of the three cysteine residues that are conserved in all A subunits sequenced thus far, two (Cys284 and Cys539) appear essential for correct folding or stability of the A subunit. Mutation of the third cysteine (Cys261), located in the glycine-rich loop, to valine, generated an enzyme that was fully active but resistant to inhibition by N-ethylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation. To test the role of disulfide bond formation in regulation of vacuolar acidification in vivo, we have also determined the effect of the C261V mutant on targeting and processing of the soluble vacuolar protein carboxypeptidase Y. No difference in carboxypeptidase Y targeting or processing is observed between the wild type and C261V mutant, suggesting that disulfide bond formation in the V-ATPase A subunit is not essential for controlling vacuolar acidification in the Golgi. In addition, fluid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in the C261V and wild type cells. Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of V-ATPase activity. The E286Q mutant showed labeling by 2-azido-[32P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.     

Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain

The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. In previous studies the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 was identified to be involved in this assembly, and the sites for beta and beta' association were suggested to be located within or near the two conserved regions in this amino-terminal assembly domain of alpha. For detailed functional mapping, Ala was substituted for 26 highly conserved amino acids around residues 40, 80 and 170 to 210. The alpha-point mutants were analyzed in vitro for their abilities to form dimers and to assemble beta beta' subunits. New types of assembly-deficient mutants were identified: alpha-R45A (having substituted Ala for Arg at residue 45) dimerized but did not assemble beta (and beta') subunits; and alpha-L48A showed a decreased level of alpha 2 beta subassembly formation, indicating that this region (residues 45 to 48) is responsible for beta-binding. Isolation of two mutants, alpha-K86A and alpha-V173A, both forming alpha 2 beta but not alpha 2 beta beta' complex, confirmed our previous conclusion that two separated regions participate in beta'-binding.     

Over expression of alpha-hANP in two kinds of expression systems of Escherichia coli

Two kinds of expression plasmids carried Ref-Adaptor-(alpha-hANP) hybrid gene have been constructed. The adaptor GAA is the codon of glutamic acid which is a recognition site of the endoproteinase Glu-C. The expression of the heterologous fusion gene was controlled by the bacteriophage lambda PL promoter in the Escherichia coli expression system which is affected by the cIts857 repressor. These overproduced proteins were found in the form of inclusion bodies and constituted 35% and 48% of the total cellular protein respectively. The fusion protein was isolated and cleaved with Glu-C to liberate mature alpha-hANP, which demonstrated significant activities for reduction of blood pressure and vasorelaxation comparable to that of the chemically synthesized hormone. In this paper, the comparison between the two kinds of expression systems is also discussed.     

Analysis of a genetic mutation in an electrophoretic variant of the human lactate dehydrogenase-B(H) subunit

An electrophoretic variant of the lactate dehydrogenase (LDH)-B(H) subunit was discovered in a patient with diabetes mellitus. His LDH activity in serum was slightly lower than normal and the LDH isozyme pattern showed an abnormal migration indicating an LDH-B subunit variant of the fast type. The LDH containing the variant subunit revealed a decreased heat stability. DNA analysis of the variant allele detected a base substitution, an A to G transition, at codon 6 (AAA-->GAA). The mutation resulted in the replacement of a lysine by a glutamic acid (K6E). The change may cause the heat instability and affect the net charge of the variant subunit, resulting in an electrophoretic LDH-B subunit variant of the fast type.