Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing alpha 1 connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic "loop" domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr approximately 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252-271. Immunoelectron microscopy demonstrated that the Mr approximately 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr approximately 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the alpha 1 polypeptide. Therefore, the Mr approximately 13,000 carboxyterminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexins indicates conservation in the molecular design of the gap junction channels formed by alpha or beta connexins.
J Mol Biol 1992 Feb 20
PMID:Membrane topology and quaternary structure of cardiac gap junction ion channels. 137 48

1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [alpha-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [alpha-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio).(ABSTRACT TRUNCATED AT 400 WORDS)
Cell Mol Neurobiol 1992 Aug
PMID:Isoprenylation and carboxylmethylation in small GTP-binding proteins of pheochromocytoma (PC-12) cells. 139 71

Formation of the asymmetrically located septum divides sporulating bacilli into two distinct cells: the mother cell and the prespore. The rigidifying wall material in the septum is subsequently removed by autolysis. Examination of published electron micrographs indicates that the two septal membranes then fuse to form a single membrane. Membrane fusion would be expected to have profound consequences for subsequent development. For example, it is suggested that fusion activates processing of pro-sigma E to sigma E in the cytoplasm by exposing it to a membrane-bound processing enzyme. Asymmetry of the fused membrane could restrict processing to one face of the membrane and hence explain why sigma E is associated with transcription in the mother cell but not in the prespore. Asymmetry of the fused membrane might also provide a mechanism for restricting the activity of another factor, sigma F, to the prespore. Attachment of the flexible fused septal membrane to the condensing prespore nucleoid could help drive the engulfment of the prespore by the mother cell.
Mol Microbiol 1992 Sep
PMID:Septal membrane fusion--a pivotal event in bacterial spore formation? 144 70

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome.
Mol Microbiol 1992 Nov
PMID:DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans. 145 53

The czcR gene, one of the two control genes responsible for induction of resistance to Co2+, Zn2+, and Cd2+ (czc system) in the Alcaligenes eutrophus plasmid pMOL30, was cloned and characterized. The 1,376-bp sequence upstream of the czcCBAD structural genes encodes a 41.4-kDa protein, the czcR gene product, transcribed in the opposite direction of that of the czcCBAD genes. The putative CzcR polypeptide (355 amino acid residues) contains 11 cysteine and 14 histidine residues which might form metal cation-binding sites. A czcC::lacZ reporter gene translational fusion was constructed, inserted into plasmid pMOL30 in A. eutrophus, and expressed under the control of CzcR. Zn2+, Co2+, and Cd2+, as well as Ni2+, Cu2+, Hg2+, and Mn2+ and even Al3+, served as inducers of beta-galactosidase activity. Besides the CzcR protein, the membrane-bound CzcD protein was essential for induction of czc. The CzcR and CzcD proteins display no sequence similarity to two-component regulatory systems of a sensor and a response activator type; however, CzcD has 34% identity with the ZRC-1 protein, which mediates zinc resistance in Saccharomyces cerevisiae (A. Kamizomo, M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura, Mol. Gen. Genet. 219:161-167, 1989).
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PMID:CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus. 145 58

Evidence is reported for the existence of a structurally and functionally related and probably evolutionarily conserved class of membrane-bound liver carbonyl reductases/hydroxysteroid dehydrogenases involved in steroid and xenobiotic carbonyl metabolism. Carbonyl reduction was investigated in liver microsomes of 8 vertebrate species, as well as in insect larvae total homogenate and in purified 3 alpha-hydroxysteroid dehydrogenase preparations of the procaryont Pseudomonas testosteroni, using the ketone compound 2-methyl-1,2 di-(3-pyridyl)-1-propanone (metyrapone) as substrate. The enzyme activities involved in the metyrapone metabolism were screened for their sensitivity to several steroids as inhibitors. In all fractions tested, steroids of the adrostane or pregnane class strongly inhibited xenobiotic carbonyl reduction, whereas only in the insect and procaryotic species could ecdysteroids inhibit this reaction. Immunoblot analysis with antibodies against the respective microsomal mouse liver metyrapone reductase revealed strong crossrections in all fractions tested, even in those of the insect and the procaryont. A similar crossreaction pattern was achieved when the same fractions were incubated with antibodies against 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The mutual immunoreactivity of the antibody species against proteins from vertebrate liver microsomes, insects and procaryonts suggests the existence of structural homologies within these carbonyl reducing enzymes. This is further confirmed by limited proteolysis of purified microsomal mouse liver carbonyl reductase and subsequent analysis of the peptide fragments with antibodies specifically purified by immunoreactivity against this respective crossreactive antigen. These immunoblot experiments revealed a 22 kDa peptide fragment which was commonly recognized by all antibodies and which might represent a conserved domain of the enzyme.
J Steroid Biochem Mol Biol 1992 Dec
PMID:Homologies between enzymes involved in steroid and xenobiotic carbonyl reduction in vertebrates, invertebrates and procaryonts. 147 59

Stimulation of cardiac phospholipid metabolism has diverse biological effects, ranging from subtle changes in cellular function to severe cellular damage. Accordingly, knowledge of the factors governing the activity of cardiac phospholipases is of great biological importance. A possible role of annexins, intracellular proteins that bind to membranes in a calcium dependent manner, as modulators of phospholipase activity has been proposed. In this study we investigated the cell type specific distribution of Annexin V and VIII in the heart. Recombinant Annexin V was used to examine the effect of this type of Annexin on cardiac phospholipase activity. Western blot analysis shows that annexin V is abundantly present in the heart. Using isolated myocytes and cultured cardiac endothelial and fibroblast-like cells, it is demonstrated that the localization of Annexin V is confined to non-myocytes. No positive bands matching the Mw of recombinant Annexin VIII are found in any of the cell types examined. In vitro studies demonstrate that recombinant Annexin V potently inhibits the activity of cardiac membrane-bound phospholipases, acting on their natural surrounding substrate, in a calcium dependent manner. Interestingly, annexin V also inhibits triacylglycerol hydrolysis. In conclusion, the expression of annexins is cell-type specific and suggests a cell-type specific function of these proteins in the heart. The absence of Annexin V in cardiac myocytes dismisses involvement of this annexin in cardiomyocyte phospholipid metabolism. The presence of Annexin V in cardiac endothelial and fibroblasts suggests a regulating role in the phospholipid homeostasis of non-myocyte cell types in the heart.
Mol Cell Biochem 1992 Oct 21
PMID:Annexins in cardiac tissue: cellular localization and effect on phospholipase activity. 148 Jan 59

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme responsible for the alpha-amidation of peptides in secretory granules of neuroendocrine cells. The single gene encoding PAM undergoes tissue-specific alternative splicing and endoproteolytic processing to generate bifunctional membrane proteins with a single transmembrane domain as well as soluble proteins that are mono- or bifunctional. In order to examine the endoproteolytic processing and subcellular localization of the various forms of PAM in cells lacking regulated secretory granules, we established stably transfected hEK-293 cell lines expressing naturally occurring and mutant forms of PAM. As expected, newly synthesized soluble PAM proteins were rapidly secreted into the medium. Integral membrane protein forms of PAM were largely localized in the perinuclear region with punctate staining visible throughout the cell and 2-5% of the enzyme activity detectable on the cell surface. Bifunctional PAM proteins were slowly released into the medium after expression of integral membrane protein forms of PAM. Deletion of 77 amino acids from the COOH-terminus of the integral membrane forms of PAM resulted in a membrane-bound protein which retained both enzymatic activities but accumulated on the cell surface. Rapid internalization of full-length PAM proteins was observed by incubating live cells with antiserum to PAM; deletion of the COOH-terminal domain eliminated the ability of cells to internalize PAM. Thus the cytoplasmic domain of integral membrane PAM contains a routing determinant recognized by cells lacking the regulated secretory pathway.
Mol Endocrinol 1992 Dec
PMID:Expression of a peptide processing enzyme in cultured cells: truncation mutants reveal a routing domain. 149 98

Citrate is fermented by Klebsiella pneumoniae to 2 acetate, 0.5 formate and 1.2 CO2. The formation of less than 1 formate and greater than 1 CO2 per citrate can be accounted for by the oxidation of formate to CO2 in order to provide reducing equivalents for the assimilation of citrate into cell carbon. A membrane-bound electron transport chain is apparently involved in NADH synthesis by these cells. The electrons from formate oxidation to CO2 are used to reduce ubiquinone to ubiquinol by membrane-bound formate dehydrogenase and ubiquinol further delivers its electrons to NAD+, if this endergonic reaction is powered by delta mu Na+. The endogenous NADH level of K. pneumoniae cells thus increased in the presence of formate in response to a delta pNa+ greater than -100 mV. NADH formation was completely abolished in the presence of oxygen or after addition of hydroxyquinoline-N-oxide, a specific inhibitor of the Na(+)-translocating NADH:ubiquinone oxidoreductase. The increase of endogenous NADH was dependent on the delta pNa+ applied to the cells. Inverted membrane vesicles of K. pneumoniae catalysed the reduction of NAD+ to NADH with formate as electron donor after application of delta mu Na+ of about 120 mV consisting of delta pNa+ of 60 mV and delta psi of the same magnitude. Neither the delta pNa+ nor the delta psi of this size alone was sufficient to drive the endergonic reaction. Strictly anaerobic conditions were required for NADH formation and hydroxyquinoline-N-oxide completely inactivated the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Microbiol 1992 Jul
PMID:NADH formation by Na(+)-coupled reversed electron transfer in Klebsiella pneumoniae. 150 43

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Jul
PMID:Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells. 150 22


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