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1.
Y Nonaka H Murakami Y Yabusaki S Kuramitsu H Kagamiyama T Yamano M Okamoto 《Biochemical and biophysical research communications》1987,145(3):1239-1247
A full-length cDNA clone (pADR) for adrenodoxin reductase was isolated by means of immunological screening from a bovine adrenal poly(A)+ mRNA library. A cDNA insert of 1,973 base pairs in length encoded the entire amino acid sequence of the adrenodoxin reductase precursor protein, which consists of 492 amino acids including an extrapeptide of 32 amino acids at the NH2-terminus. The cloned cDNA contained the complete 3'-noncoding region of 443 nucleotides including 59 nucleotides of poly(A) and 51 nucleotides in the 5'-noncoding region. The amino acid sequences from the 33rd to 70th, the 117th to 123rd, the 207th to 225th, the 247th to 323rd, the 385th to 426th, the 444th to 461st, and the 487th to 492nd in the predicted structure were identical with those of the purified adrenodoxin reductase and its digested peptides, with only four exceptions. 相似文献
2.
When rat liver mitochondria that had imported a synthetic extrapeptide of ornithine aminotransferase (composed of 34 amino acids) were incubated at 25 degrees C, the extrapeptide in their matrix was degraded inside the mitochondria. The degradation of the extrapeptide did not depend on energy either inside or outside the mitochondria. The degrading activity was found exclusively in the mitochondrial soluble fraction and only inhibited by N-ethylmaleimide of eight protease-inhibitors tested. These observations show that the extrapeptide cleaved from the precursor of the mitochondrial protein in the mitochondria is degraded by some ATP-independent proteases inside the mitochondria. 相似文献
3.
The cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria 总被引:13,自引:0,他引:13
The mechanism of import of proteins into mitochondria was studied by using the peptide of the presequence of ornithine aminotransferase (the extrapeptide), which was chemically synthesized and is composed of 34 amino acids. When the extrapeptide was incubated with isolated mitochondria in the presence of a rabbit reticulocyte lysate at 25 degrees C, it was imported into the mitochondrial matrix, and the import depended on the inner membrane potential, but not added ATP. The import of several precursors of mitochondrial proteins was competitively inhibited by the presence of excess extrapeptide in the reaction system, indicating that the extrapeptide and mitochondrial proteins were imported by the same machinery. Import of the extrapeptide was significantly stimulated by addition of a rabbit reticulocyte lysate, and a component of the lysate (the cytosolic factor) stimulating import of the extrapeptide was purified about 20,000 times by successive column chromatography on DEAE-cellulose and aminopentyl-Sepharose 4B. The binding of the extrapeptide to liposomes composed of egg lecithin and partially purified receptor of the precursor of mitochondrial protein (Ono, H., and Tuboi, S., (1985) Biochem. Int. 10, 351-357) required the cytosolic factor when the concentration of the peptide was less than 1.5 X 10(-8) M, suggesting that the physiological binding of the precursors of mitochondrial proteins to the receptor is dependent on the cytosolic factor. The extrapeptide and the cytosolic factor were shown to form a complex. From these results, the mechanism of binding of the extrapeptide to the receptor of the mitochondrial outer membrane is suggested to be as follows: the peptide (the precursor of mitochondrial protein) and the cytosolic factor form a complex, and then the complex is recognized by and bound to the receptor. 相似文献
4.
Synthetic partial extension peptides of P-450(SCC) and adrenodoxin precursors: effects on the import of mitochondrial enzyme precursors 总被引:4,自引:0,他引:4
S Furuya M Okada A Ito H Aoyagi T Kanmera T Kato Y Sagara T Horiuchi T Omura 《Journal of biochemistry》1987,102(4):821-832
Various portions of the extension peptides of P-450(SCC) precursor were chemically synthesized. The effects of these peptides on the import of enzyme precursors into mitochondria were examined. Peptides SEP1-15 and SEP1-20, corresponding to the amino terminal portion of the extension peptides, strongly inhibited the import of P-450(SCC) precursor into mitochondria. These peptides were effective at concentrations above 30 microM, and complete inhibition was observed at 100 microM. SEP1-11, which is shorter than SEP1-15 and SEP1-20, showed very weak inhibition. SEP25-39, which corresponds to the carboxy terminal portion of the extension peptide, did not affect the import of the precursor. The import of P-450(11 beta) and adrenodoxin precursors were also inhibited by SEP1-15. Another peptide, AEP1-14, which corresponds to the amino terminal portion of the extension peptide of adrenodoxin precursor, was also synthesized. The peptide inhibited the import of both adrenodoxin and P-450(SCC) precursors into mitochondria. The import of malate dehydrogenase was also inhibited by SEP1-15 and AEP1-14. The rate of the internalization of the precursor into mitochondria was decreased by the peptides. The amount of the precursor bound to the surface of mitochondria and the processing of adrenodoxin precursor were not affected. The respiratory activities of isolated mitochondria were not influenced by SEP1-15 even at 100 microM. We conclude that the inhibitory activities of the synthetic partial extension peptides on the import of enzyme precursors into mitochondria require the presence of about fifteen amino acid residues in the amino terminal portion of the extension peptides, and the inhibition of the import by the peptides was dependent on the blockage of the internalization of the precursors into mitochondria. 相似文献
5.
Critical region in the extension peptide for the import of cytochrome P-450(SCC) precursor into mitochondria 总被引:1,自引:0,他引:1
In order to establish the role of the extension peptide of the precursor of P-450(SCC), a mitochondrial inner membrane protein, in the import into the organella, three deletion mutants of the precursor, in which the deletions were in the mature portion, were constructed. These mutant precursors were imported into mitochondria in vitro as efficiently as the original precursor, indicating that the extension peptide contains sufficient information for the import of the precursor into mitochondria. To investigate which portion of the extension peptide contains the mitochondrial targeting signal, various lengths of the amino-terminal portion of the extension peptide of P-450(SCC) precursor were fused to the mature portion of adrenodoxin. The fusion proteins consisting of 44 and 19 amino-terminal amino acids and mature adrenodoxin were imported into mitochondria, whereas those containing 14, 7, and 2 amino-terminal amino acid residues were not. The importance of the amino-terminal portion of the extension peptide was confirmed by the deletion from the amino-terminal end of a fusion protein consisting of the amino-terminal 44 amino acid residues of P-450(SCC) precursor and mature adrenodoxin, SCC44RAd. The amino-terminal deletions abolished the import of the fusion proteins into mitochondria. Substitution of all of the three basic amino acids, Arg(4), Arg(9), and Lys(14) in the extension peptide of SCC44RAd to Ser or Thr inhibited the binding of the fusion protein to mitochondria as well as its import.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import 总被引:6,自引:0,他引:6
The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import. 相似文献
7.
Proteinase-polymerase precursor as the active form of feline calicivirus RNA-dependent RNA polymerase 总被引:2,自引:0,他引:2
Wei L Huhn JS Mory A Pathak HB Sosnovtsev SV Green KY Cameron CE 《Journal of virology》2001,75(3):1211-1219
The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a threefold reduction in polymerase activity. Deletion of an additional one (the Thr-1236 amino terminus) or two (the Ala-1237 amino terminus) amino acids produced derivatives that were 7- and 175-fold, respectively, less active than Pro-Pol. FCV proteinase-dependent processing of Pro-Pol in the interdomain region preceding Val-1235 was not observed in the presence of a catalytically active proteinase; however, processing within the polymerase domain was observed. Inactivation of proteinase activity by changing the catalytic cysteine-1193 to glycine permitted the production and purification of intact Pro-Pol. Biochemical analysis of Pro-Pol showed that this enzyme has properties expected of a replicative polymerase, suggesting that Pro-Pol is an active form of the FCV RdRP. 相似文献
8.
A 15-kDa interferon-induced protein is derived by COOH-terminal processing of a 17-kDa precursor 总被引:7,自引:0,他引:7
E Knight D Fahey B Cordova M Hillman R Kutny N Reich D Blomstrom 《The Journal of biological chemistry》1988,263(10):4520-4522
An interferon-induced 15-kDa protein is synthesized from a precursor of higher molecular weight; the precursor contains 165 amino acids (17 kDa), whereas the stable product (15 kDa) contains 156 amino acids. The stable 15-kDa form is derived from the precursor 17-kDa form by the removal of eight amino acids from the COOH terminus and the methionine from the NH2 terminus. The existence of the precursor 17-kDa protein can be demonstrated after brief periods of in vivo labeling with [35S]methionine and by translation of mRNA in vitro. 相似文献
9.
10.
Glyoxysomal Malate Dehydrogenase in Pumpkin: Cloning of a cDNA and Functional Analysis of Its Presequence 总被引:3,自引:0,他引:3
Kato Akira; Takeda-Yoshikawa Yoko; Hayashi Makoto; Kondo Maki; Hara-Nishimura Ikuko; Nishimura Mikio 《Plant & cell physiology》1998,39(2):186-195
Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of theglyoxylate cycle that participates in degradation of storageoil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledonsthat encodes a polypep-tide consisting of 356 amino acid residues.The nucleotide and N-terminal amino acid sequences revealedthat gMDH is synthesized as a precursor with an N-terminal extrapeptide.The N-terminal presequence of 36 amino acid residues containstwo regions homologous to those of other micro-body proteins,which are also synthesized as large precursors. To investigatethe functions of the N-terminal presequence of gMDH, we generatedtransgenic Arabidopsis that expressed a chimeric protein consistingof rß-glucuroni-dase and the N-terminal region ofgMDH. Immunologi-cal and immunocytochemical studies revealedthat the chimeric protein was imported into microbodies suchas gly-oxysomes and leaf peroxisomes and was then subsequentlyprocessed. Site-directed mutagenesis studies showed that theconserved amino acids in the N-terminal presequence, Arg-10and His-17, function as recognition sites for the targetingto plant microbodies, and Cys-36 in the presequence is responsiblefor its processing. These results correspond to those from theanalyses of glyoxysomal citrate synthase (gCS), which was alsosynthesized as a large precursor, suggesting that common mechanismsthat can recognize the targeting or the processing of gMDH andgCS function in higher plant cells. (Received July 10, 1997; Accepted November 22, 1997) 相似文献
11.
We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR). The precursor of rat AdR was predicted to consist of 34 amino-terminal residues of extrapeptide for transport into mitochondria and the following 460 residues of the mature peptide region. The deduced amino acid sequence was 70.8 and 61.8% homologous to those of bovine and human AdRs in the extrapeptide region, respectively, and 88.5% homologous to both the sequences of bovine and human AdRs in the mature peptide region. The predicted mature form of rat AdR was directly expressed in Escherichia coli, using cDNA, and was purified with a yield of 32 mg/l of culture. The purified recombinant rat AdR showed an absorption spectrum characteristic of a flavoprotein with peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm. The extinction coefficient was estimated to be 10.9 mM(-1) cm(-1) at 450 nm. The absorbance ratio at 270 nm/450 nm was 7.1. From the θ(208) value in the circular dichroism spectrum, the alpha-helix content in the rat AdR was calculated to be 30%. In NADPH-cytochrome c reductase activity reconstituted with adrenodoxin (Ad), the apparent K(m) value of rat AdR for NADPH was 0.32 microM, a value significantly lower than that of bovine AdR (1.4 microM). The rat AdR showed a higher affinity to the heterologous redox partner (bovine Ad, K(m)=9.3 nM) than to the native partner (rat Ad, K(m)=16.7 nM), whereas the affinity of bovine AdR was slightly higher to the native partner (bovine Ad, K(m)=37.1 nM) than to the heterologous partner (rat Ad, K(m)=46.8 nM). The K(m) values showed a reverse correlation to the difference of pI values between the redox partners. These results indicate that AdR binds to Ad mainly by ionic interaction. 相似文献
12.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1995,1246(1):39-46
Previously, we have proposed that bovine adrenocortical mitochondrial adrenodoxin reductase may possess a domain structure, based upon the generation of two major peptide fragments from limited tryptic proteolysis. In the present study, kinetic characterization of the NADPH-dependent ferricyanide reductase activity of the partially proteolyzed enzyme demonstrates that Km(NADPH) increases (from 1.2 μM to 2.7 μM), whereas 1 Vmax remains unaltered at 2100 min−1 The two proteolytic fragments have been purified to homogeneity by reverse-phase HPLC, and amino acid sequence analysis unambiguously demonstrates that the 30.6 kDa fragment corresponds to the amino terminal portion of the intact protein, whereas the 22.8 kDa fragment is derived from the carboxyl terminus of the reductase. Trypsin cleavage occurs at either Arg-264 or Arg-265. Covalent crosslinking experiments using a water-soluble carbodiimide show that adrenodoxin crosslinks exclusively to the 30.6 kDa fragment, thus implicating the N-terminal region of adrenodoxin reductase in binding to the iron-sulfur protein. Our inability to detect covalent carbohydrate on either intact or proteolyzed adrenodoxin reductase prompted a re-examination of the previously reported requirement of an oligosaccharide moiety for efficient electron transfer from the reductase to adrenodoxin. Treatment of adrenodoxin reductase with a highly purified preparation of neuraminidase demonstrates that neither the adrenodoxin-independent ferric yanide reductase activity nor the adrenodoxin-dependent cytochrome c reductase activity of the enzyme is affected by neuraminidase treatment. 相似文献
13.
Recombinant HIV2 protease processes HIV1 Pr53gag and analogous junction peptides in vitro 总被引:5,自引:0,他引:5
S Pichuantes L M Babé P J Barr D L DeCamp C S Craik 《The Journal of biological chemistry》1990,265(23):13890-13898
14.
J L Millán 《The Journal of biological chemistry》1986,261(7):3112-3115
The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced. 相似文献
15.
Characterization of two allelic forms of Neurospora crassa laccase. Amino- and carboxyl-terminal processing of a precursor 总被引:22,自引:0,他引:22
The complete structures of the laccase genes isolated from two different Neurospora crassa wild-type strains are described. The genes were cloned by screening partial genomic DNA libraries with a nick-translated laccase-specified 1.36-kilobase SalI fragment (Germann, U. A., and Lerch, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8854-8858) as a hybridization probe. Nucleotide sequence analysis revealed the presence of two different allelic forms. They conform to the same structural organization, but show an overall divergence of 5.3% which is mainly the result of point mutations in the nontranslated regions. The coding parts are interrupted by a short intron. The encoded proteins differ in 12 out of 619 amino acid residues. A comparison of the primary structure deduced from the nucleotide sequence of the gene with a protein chemical analysis of the two terminal cyanogen bromide fragments of extracellular N. crassa laccase revealed that the enzyme is synthesized as a precursor. The precursor protein exceeds the mature protein by 49 amino acids at its amino terminus and by 13 amino acids at its carboxyl terminus, thus indicating a complex maturation pathway. The possible involvement of amino-terminal processing in secretion and of carboxyl-terminal processing in activation of the enzyme is discussed. 相似文献
16.
Jack Lenz Robert Crowther Anthony Straceski William Haseltine 《Journal of virology》1982,42(2):519-529
The sequence of 2,191 nucleotides encoding the env gene of murine retrovirus Akv was determined by using a molecular clone of the Akv provirus. Deduction of the encoded amino acid sequence showed that a single open reading frame encodes a 638-amino acid precursor to gp70 and p15E. In addition, there is a typical leader sequence preceding the amino terminus of gp70. The locations of potential glycosylation sites and other structural features indicate that the entire gp70 molecule and most of p15E are located on the outer side of the membrane. Internal cleavage of the env precursor to generate gp70 and p15E occurs immediately adjacent to several basic amino acids at the carboxyl terminus of gp70. This cleavage generates a region of 42 uncharged, relatively hydrophobic amino acids at the amino terminus of p15E, which is located in a position analogous to the hydrophobic membrane fusion sequence of influenza virus hemagglutinin. The mature polypeptides are predicted to associate with the membrane via a region of 30 uncharged, mostly hydrophobic amino acids located near the carboxyl terminus of p15E. Distal to this membrane association region is a sequence of 35 amino acids at the carboxyl terminus of the env precursor, which is predicted to be located on the inner side of the membrane. By analogy to Moloney murine leukemia virus, a proteolytic cleavage in this region removes the terminal 19 amino acids, thus generating the carboxyl terminus of p15E. This leaves 15 amino acids at the carboxyl terminus of p15E on the inner side of the membrane in a position to interact with virion cores during budding. The precise location and order of the large RNase T(1)-resistant oligonucleotides in the env region were determined and compared with those from several leukemogenic viruses of AKR origin. This permitted a determination of how the differences in the leukemogenic viruses affect the primary structure of the env gene products. 相似文献
17.
Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15 总被引:3,自引:0,他引:3
A Amemura R Chakraborty M Fujita T Noumi M Futai 《The Journal of biological chemistry》1988,263(19):9271-9275
The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned. Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase. The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes. These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes. Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase. The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase. 相似文献
18.
The conformations of synthetic peptides which span the region in which the precursor part of proteins (signal sequences) destined for export are cleaved by signal peptidases, were investigated by circular dichroism spectroscopy. Pentapeptides comprising amino acids only from the carboxy-terminus of signal sequences or the amino terminus of the mature protein do not have any preferred conformation in a variety of solvents. Octa- and nonapeptides containing amino acids from the carboxy-terminal protion of signal sequences and the amino-terminus of the mature portions of precursor proteins tend to adopt beta-turn conformations in trifluoroethanol and micelles of sodium dodecylsulphate. Hence, in addition to the distribution of amino acids with small side chains at the carboxy terminus of signal sequences, it is conceivable that signal peptidases also recognize a beta-turn conformation in the cleavage site region of precursor proteins. 相似文献
19.
Certain general principles determine the biosynthesis of most biologically active peptides, including the opioid peptides, from large protein precursors. In almost all instances, the active peptide is embedded in the precursor flanked on both sides by pairs of basic amino acids. The first step in processing involves a trypsinlike enzyme, cleaving to the carboxyl terminus of basic amino acids, and leaving the active peptide with a basic amino acid on the carboxyl terminus. A carboxy-peptidase peptidase B-like enzyme then removes the remaining basic amino acid. It has been unclear whether any endopeptidases with trypsinlike activity are selective for one or another basic amino acid. Recently a soluble endopeptidase has been identified that can cleave to both the carboxyl and amino termini of basic amino acids. Enkephalin convertase (carboxypeptidase E, H) (EC 3.4.17.10) has considerable selectivity, and appears to be physiologically associated with the biosynthesis of enkephalin as well as a limited number of other neuropeptides. The turnover of opioid peptides and other neuropeptides is most effectively ascertained by measuring levels of mRNA either biochemically or by in situ hybridization. Striking dynamic alterations include a pronounced increase in levels of proenkephalin mRNA in the corpus striatum after blockade of dopamine receptors, but changes in opioid peptide mRNA after opiate addiction are less clear. 相似文献