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1.
Myosin light chain phosphatase consists of three subunits, a 38-kDa catalytic subunit, a large 110-130-kDa myosin binding subunit, and a small subunit of 20-21 kDa. The catalytic subunit and the large subunit have been well characterized. The small subunit has been cloned and studied from smooth muscle, but little is known about its function and specificity in the other muscles such as cardiac muscle. In this study, cDNAs for heart-specific small subunit isoforms, hHS-M(21), were isolated and characterized. Evidence was obtained from an analysis of genome to suggest that the small subunit was the product of the same gene as the large subunit. Using permeabilized renal artery preparation and permeabilized cardiac myocytes, it was shown that the small subunit increased sensitivity to Ca(2+) in muscle contraction. It was also shown using an overlay assay that hHS-M(21) bound the large subunit. Mapping experiments demonstrated that the binding domain and the domain involved in the increasing Ca(2+) sensitivity mapped to the same N-terminal region of hHS-M(21). These observations suggest that the heart-specific small subunit hHS-M(21) plays a regulatory role in cardiac muscle contraction by its binding to the large subunit.  相似文献   

2.
Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. Each subunit of the G protein complex is encoded by a member of one of three corresponding gene families. Currently, 16 different members of the alpha subunit family, 5 different members of the beta subunit family, and 11 different members of the gamma subunit family have been described in mammals. Here we have identified and characterized Bacterial Artificial Chromosomes (BACs) containing the human homologs of each of the alpha, beta, and gamma subunit genes as well as a G alpha11 pseudogene and a previously undiscovered G gamma5-like gene. The gene structure and chromosome location of each gene was determined, as were the orientations of paired genes. These results provide greater insight into the evolution and functional diversity of the mammalian G protein subunit genes.  相似文献   

3.
We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase beta subunit. A bovine abomasum lambda gt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase beta subunit. A single positive phage clone containing an approximately 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase beta subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase beta subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length beta subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase beta subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase beta 2 subunit and shares a number of structural similarities with Na,K-ATPase beta subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase beta subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase beta subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.  相似文献   

4.
K Kondo  T Beppu    S Horinouchi 《Journal of bacteriology》1995,177(17):5048-5055
The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.  相似文献   

5.
Pertussis toxin and target eukaryotic cells: binding, entry, and activation.   总被引:20,自引:0,他引:20  
H R Kaslow  D L Burns 《FASEB journal》1992,6(9):2684-2690
Pertussis toxin, a protein virulence factor produced by Bordetella pertussis, is composed of an A protomer and a B oligomer. The A protomer consists of a single polypeptide, termed the S1 subunit, which disrupts transmembrane signaling by ADP-ribosylating eukaryotic G-proteins. The B oligomer, containing five polypeptides, binds to cell receptors (most likely containing carbohydrate) and delivers the S1 subunit. Current knowledge suggests that expression of ADP-ribosyltransferase activity in target eukaryotic cells arises after 1) nucleotides and membrane lipids allosterically promote the release of the S1 subunit; and 2) the single disulfide bond in the S1 subunit is reduced by reductants such as glutathione. This model suggests conditions for the proper use of the toxin as an experimental reagent.  相似文献   

6.
We have compared the hydrodynamic shape, conformation, and stabilities of active, unwashed ribosomal subunits, as well as their susceptibilities to changes in temperature and ionic strength. Both intrinsic viscosity and sedimentation velocity measurements indicate that the 30 S subunit has a more asymmetric hydrodynamic shape. The intrinsic viscosity of this subunit in reconstitution buffer has been found to be significantly larger than the value reported previously. While the RNA conformation in both subunits may be very similar as suggested by the near uv CD spectra, the average conformation of the protein in the two subunits is drastically different. The 30 S subunit has a lower Tm. The 50 S subunit is rather stable toward changes of ionic strength, whereas the 30 S subunit is quite susceptible to changes in ionic strength.  相似文献   

7.
Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four alpha-type and two beta-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophila alpha subunits are co-expressed with vertebrate beta subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned beta-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR beta-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dbeta3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dbeta3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells.  相似文献   

8.
Mac-1 (CD 11b/CD18) is a leukocyte adhesion heterodimeric glycoprotein which functions both as a receptor for iC3b (CR3) and in several cell-cell and cell-substrate adhesive interactions. We describe full-length cDNA clones for the alpha subunit of Mac-1. Mac-1 alpha subunit message was detected in blood monocytes and phorbol-12-myristate acetate-induced myeloid cell lines, but not in cells of the T or B lineages, correlating with Mac-1 protein surface expression. The alpha subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail. The extracellular domain contains three putative divalent cation-binding sequences and 19 potential N-glycosylation sites. The amino acid sequence of Mac-1 alpha shows that it is a member of the integrin superfamily; Mac-1 alpha shows 63% identity to the alpha subunit of the leukocyte adhesion glycoprotein p150.95 and 25% to the alpha subunits of the extracellular matrix receptors platelet glycoprotein IIb/IIIa, the fibronectin receptor, and the vitronectin receptor. The Mac-1 alpha subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree of identity both to the p150.95 alpha subunit (87% identity at the amino acid level) and to the rest of the integrin alpha subunits (38%). The alpha subunit of Mac-1, like the p150.95 alpha subunit, contains a domain of 187 amino acids in the extracellular region which is absent in other integrins. This leukocyte or "L" domain is homologous to the A domains of von Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2. These findings draw attention to this region of Mac-1 as a potential binding site for iC3b.  相似文献   

9.
MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.  相似文献   

10.
The B subunit of cholera toxin, which binds specifically to ganglioside GM1, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts grown in chemically defined medium. The mitogenic response to the B subunit was potentiated by insulin and other growth factors. To elucidate the mechanism by which the B subunit stimulates cell growth , its effects on several transmembrane signaling systems which have been suggested to play a vital role in cell growth regulation were examined. The B subunit did not increase cAMP levels nor activate adenylate cyclase. The B subunit induced a rapid and profound increase in intracellular free Ca2+ as measured with the fluorescent Ca2+-sensitive dye quin 2/AM. Removal of external Ca2+ completely inhibited the signal, thus suggesting that the B subunit elevates intracellular Ca2+ through a net influx of extracellular Ca2+ rather than by causing the release of Ca2+ from intracellular stores. These findings are consistent with the observations that the B subunit induced reinitiation of DNA synthesis without activation of phospholipase C. There was no increase in the formation of inositol trisphosphate, the second messenger that mediates release of Ca2+ from intracellular stores. In addition, the B subunit still stimulated DNA synthesis in Swiss 3T3 cells pretreated with phorbol ester to down-regulate protein kinase C. These results suggest that the mitogenic effects of the B subunit are mediated mainly by facilitation of Ca2+ influx and that activations of adenylate cyclase, phospholipase C, or protein kinase C are not obligatory steps in the initiation of cell growth by the B subunit. Furthermore, the observation that Ca2+ ionophores, such as ionomycin and A23187, are not mitogenic implies that additional undefined growth signaling pathways may exist in this system.  相似文献   

11.
The circularly permuted GTPase YlqF is essential for cell viability and is broadly conserved from Gram-positive bacteria to eukaryotes. We previously reported that YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis. Here, we demonstrate that an N-terminal deletion mutant of YlqF (YlqFDeltaN10) inhibits cell growth even in the presence of wild-type YlqF. In contrast to the wild-type protein, the GTPase activity of this mutant was not stimulated by the 50 S subunit and did not dissociate from the premature 50 S subunit. Thus, YlqFDeltaN10 acts as a competitive inhibitor of wild-type YlqF. Premature 50 S subunit lacking ribosomal protein L27 and with a reduced amount of L16 accumulated in YlqFDeltaN10-overexpressing cells and in YlqF-depleted cells, suggesting that YlqFDeltaN10 binds to the premature 50 S subunit. Moreover, premature 50 S subunit from both YlqFDeltaN10-overexpressing and YlqF-depleted cells more strongly enhanced the GTPase activity of YlqF than the mature 50 S subunit of the 70 S ribosome. Collectively, our results indicate that YlqF is targeted to the premature 50 S subunit lacking ribosomal proteins L16 and L27 to assemble functional 50 S subunit through a GTPase activity-dependent conformational change of 23 S rRNA.  相似文献   

12.
The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA.  相似文献   

13.
Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68,993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme.  相似文献   

14.
Tetranitromethane reaction with intact ovine lutropin and its isolated subunits was studied using spectrophotometric measurements, amino acid analysis, and isolation of tyrosyl peptides. Tyrosyl residues in the beta subunit (beta37, beta59) did not react with tetranitromethane in the intact hormone, but were nitrated in the isolated subunit. The sequence and extent of reaction of tetranitromethane with the tyrosyl residues in the alpha subunit was alpha21 = alpha92 = alpha93 (in intact hormone or isolated subunit) greater than alpha 41 (reacted in isolated subunit only) greater than alpha 30 (reacted in isolated subunit in 8 M urea only). Polymerization was observed as a side reaction in agreement with previous studies. The degree of polymerization appeared to be related to both primary sequence and tertiary structure, and for lutropin had the relation: alpha subunit (93% polymerized) greater than intact hormone greater than beta subunit (less than 40%). Polymerization observed with vasopressin was significantly greater than with oxytocin; for these peptides the tyrosine residues in the monomeric product were converted to 3-nitrotyrosine. Neither 3-nitrotyrosine nor tyrosine was detected in the polymerized by-products. In the tetranitromethane reaction with intact ovine lutropin, other reaction products charcterized by absorption spectra were found. Peptides isolated from these products lacked the characteristic 428 nm abosrption maxima of 3-nitrotyrosyl peptides and showed instead absorption in the 310 to 350 nm region. Similar products from tetranitromethane reactions with di- and tripeptides containing tyrosine have been observed previously (Boyd, N.D., and Smith, D.B. (1971) Can. J. Biochem, 49, 154-161), but they have not been studied in proteins. A possible relationship to the polymerization side reaction is suggested.  相似文献   

15.
The C subunit of Ideonella dechloratans chlorate reductase has been expressed in Escherichia coli as a GST fusion protein. Purification from inclusion bodies, followed by refolding and reconstitution with heme, produced a protein with a heme/protein ratio of 0.4, and with UV-vis spectral characteristics similar to those of native chlorate reductase. Wavelength maxima for the alpha and beta bands in the reduced state were 559 and 529 nm for both native chlorate reductase and the reconstituted recombinant subunit, whereas the reduced Soret bands were found at 426 and 424 nm, respectively. These results support the notion of the C subunit as the cytochrome b moiety of I. dechloratans chlorate reductase. Moreover, the availability of a recombinant version of the C subunit is expected to facilitate further studies of electron transfer and protein interaction included in the reaction catalyzed by chlorate reductase.  相似文献   

16.
We obtained cDNA clones for cytochrome oxidase subunits IV, V, VI, and possibly VII by constructing a lambda gt11 library of Neurospora crassa cDNA and probing it with antiserum directed against Neurospora cytochrome oxidase holoenzyme. Positive clones were further characterized with antisera directed against individual cytochrome oxidase subunits and subsequently by DNA sequencing. The clones for subunits IV and V encode proteins with regions matching the known N-terminal amino acid sequences of purified Neurospora cytochrome oxidase subunits IV and V, respectively. The sequences of these clones provide the first evidence that cytochrome oxidase subunits IV and V are made as precursors with N-terminal extensions in Neurospora. The N-terminal extensions encoded by these clones share homology, and are rich in arginine, as are signal sequences of other mitochondrially destined proteins. The subunit VI clone codes for the carboxyl terminus of a protein homologous to the carboxy termini of yeast cytochrome oxidase subunit VI and bovine cytochrome oxidase subunit Va. The subunit VII clone contains an open reading frame for a 47-residue protein, the expected size for subunit VII. However, the protein coded by this clone has an unusual amino acid composition. Whether this clone represents an authentic cytochrome oxidase subunit is not established.  相似文献   

17.
The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.  相似文献   

18.
The stimulatory guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase is activated by exposure to guanine nucleotide analogs or to Al3+ + F-. Activated G/F can reconstitute adenylate cyclase activity when mixed with the catalytic moiety of the enzyme system in the absence of an effective free concentration of stimulatory ligand. Activation is explained by dissociation of the alpha (45,000-Da) and beta (35,000-Da) subunits of G/F. The beta subunit of G/F facilitates reversal of the activated state of the regulatory protein. This phenomenon, which has been exploited as an assay for the resolved beta subunit, has the following properties. 1) Addition of the resolved beta subunit to fluoride-activated G/F increases the initial rate of deactivation from a t 1/2 of 10 min to less than 0.5 min. 2) The enhancement of the rate of deactivation is a saturable process with a K 1/2 value of 60 ng/ml (approximately 2 nM). 3) G/F does not display beta subunit activity unless the alpha subunit has been inactivated or the subunits have been resolved. beta Subunit activity is measurable in detergent extracts of rabbit liver membranes or plasma membranes from S49 cell clones. The activity in such extracts is similar to that found with purified G/F, in that incubation at 30 degrees C in the presence of Mg2+ is required for its expression. However, cyc-, UNC, and H21a (S49 cell mutants with deficient or altered G/F activity) have amounts of beta subunit activity similar to that found in wild type S49 cells. Furthermore, the amount of beta subunit activity exceeds by 5- to 10-fold the amount expected based on the quantity of G/F in wild type extracts. All of the beta subunit activity in detergent extracts of liver membranes can be purified as a 35,000-Da polypeptide that is indistinguishable from the beta subunit of G/F. The beta subunit activity in extracts of cyc- membranes is expressed after incubation with guanine nucleotide analogs, implying association of the beta subunit with a GTP-binding protein. By analysis of the chromatographic behavior of G/F and the recently identified 41,000/35,000-Da heterodimeric substrate for the islet-activating protein from Bordetella pertussis, we have identified the 41,000-Da subunit of the substrate for islet-activating protein as the GTP-binding protein with which the majority of the beta subunit activity associates. These data have direct bearing on the mechanisms of hormonal activation and inhibition of adenylate cyclase.  相似文献   

19.
Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including okadaic acid (OA) and microcystins, specifically inhibit PP2A. To obtain biologically active recombinant PP2A and to compare the properties of the PP2A catalytic subunit alpha and beta isoforms, we expressed human PP2Acalpha and cbeta in High Five insect cells. The recombinant PP2Acalpha and cbeta possess similar phosphatase activities using p-NPP and phosphopeptide as substrates and are strongly inhibited by OA and microcystin-LR to similar degrees. In addition, PP2Acalpha or cbeta was co-expressed with PR65/A and co-purified as a core dimer, PP2AD (Aalpha/calpha and Aalpha/cbeta) with PR65alpha/Aalpha. The recombinant PP2AD bound to the B subunit in vitro. These results show that the recombinant PP2Acalpha and cbeta are identical in their ability to associate with the A and B subunits, in their phosphatase activities, and in carboxyl-methylation. Furthermore, our results show that High Five insect cells can produce biologically active recombinant PP2A, which should be a valuable tool for detecting natural toxins and investigating the mechanism of PP2A catalysis and other protein interactions.  相似文献   

20.
The gene COX VII coding for yeast cytochrome c oxidase subunit VII has been cloned by a two-step procedure. Two degenerate oligonucleotides corresponding to amino- and carboxyl-terminal protein segments were used in a polymerase chain reaction for the amplification of a major portion of subunit VII (residues 1-52), which was then used for the cloning of complete COX VII. From the nucleotide sequence, an additional amino-terminal and two additional carboxyl-terminal amino acids are predicted as compared with the described primary sequence (Power, S. D., Lochrie, M. A., and Poyton, R. O. (1986) J. Biol. Chem. 261, 9206-9209). Beside subunit VIIa the subunit described here is the only nuclear encoded subunit of cytochrome c oxidase in yeast without a leader sequence. COX VII exists as a single copy per haploid genome as shown by Southern blot and gene disruption. Null mutants produced by gene disruption at the COX VII locus were respiratory-deficient. No cytochrome c oxidase activity was detectable nor was there an assembly of the oxidase complex.  相似文献   

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