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《Bioscience, biotechnology, and biochemistry》2013,77(3):702-705
The autoproteolytic processing of mature aspartic proteinase from sunflower seeds was investigated. The mature aspartic proteinase (48 kDa) was processed at N65s-D66s in the plant-specific region of the enzyme to form 34-kDa and 14-kDa subunits. The next step was the hydrolysis of the A25s-Q26s and N97s-E98s bonds to form a 39-kDa enzyme that consisted of 29-kDa and 9-kDa disulfide-bonded subunits. Finally, bonds including V1s-M2s, M2s-S3s, C100s-D101s, and D101s-R102s were cleaved to form non-covalently bound subunits (29 kDa and 9 kDa) by eliminating the disulfide bonds in the plant-specific region of the protein. 相似文献
3.
Park H Kusakabe I Sakakibara Y Kobayashi H 《Bioscience, biotechnology, and biochemistry》2001,65(3):702-705
The autoproteolytic processing of mature aspartic proteinase from sunflower seeds was investigated. The mature aspartic proteinase (48 kDa) was processed at N65s-D66s in the plant-specific region of the enzyme to form 34-kDa and 14-kDa subunits. The next step was the hydrolysis of the A25s-Q26s and N97s-E98s bonds to form a 39-kDa enzyme that consisted of 29-kDa and 9-kDa disulfide-bonded subunits. Finally, bonds including V1s-M2s, M2s-S3s, C100s-D101s, and D101s-R102s were cleaved to form non-covalently bound subunits (29 kDa and 9 kDa) by eliminating the disulfide bonds in the plant-specific region of the protein. 相似文献
4.
Cawley NX Chino M Maldonado A Rodriguez YM Loh YP Ellman JA 《The Journal of biological chemistry》2003,278(8):5523-5530
The potent peptidic inhibitor, Y1, of the basic residue-specific yeast aspartyl protease, yapsin 1, was synthesized and characterized. The inhibitor was based on the peptide sequence of a cholecystokinin(13-33) analog that yapsin 1 cleaved with an efficiency of 5.2 x 10(5) m(-1) s(-1) (Olsen, V., Guruprasad, K., Cawley, N. X., Chen, H. C., Blundell, T. L., and Loh, Y. P. (1998) Biochemistry 37, 2768-2777). The apparent K(i) of Y1 for the inhibition of yapsin 1 was determined to be 64.5 nm, and the mechanism is competitive. Y2 was also developed as an analog of Y1 for coupling to agarose beads. The resulting inhibitor-coupled agarose beads were successfully used to purify yapsin 1 to apparent homogeneity from conditioned medium of a yeast expression system. Utilization of this new reagent greatly facilitates the purification of yapsin 1 and should also enable the identification of new yapsin-like enzymes from mammalian and nonmammalian sources. In this regard, Y1 also efficiently inhibited Sap9p, a secreted aspartyl protease from the human pathogen, Candida albicans, which has specificity for basic residues similar to yapsin 1 and might provide the basis for the prevention or control of its virulence. A single-step purification of Sap9p from conditioned medium was also accomplished with the inhibitor column. N-terminal amino acid sequence analysis yielded two sequences indicating that Sap9p is composed of two subunits, designated here as alpha and beta, similar to yapsin 1. 相似文献
5.
Yeast cell wall assembly is a highly regulated and dynamic process. A class of cell surface aspartic peptidases anchored by a glycosylphosphatidylinositol (GPI) group, collectively known as yapsins, was proposed to be involved in cell wall construction. The Saccharomyces cerevisiae Yps1p, the prototypal yapsin, is processed internally within a loop region to produce an α/β two-subunit enzyme. Here we investigated the activation mechanism of GPI-anchored Yps1p and identified some of its substrates. We report that all activation steps of GPI-Yps1p take place at the cell surface and are regulated by the environmental pH. GPI-Yps1p is active in vivo at pH 6.0 and pH 3.0 and functions as a sheddase for a subset of GPI-anchored enzymes, including itself and the Gas1 glucanosyltransferase. Importantly, while native GPI-Yps1p weakly suppresses many phenotypes associated with the yeast kex2 Δ mutant, loop mutants that interfere with conversion into the two-subunit enzyme restore the kex2 Δ phenotypes to near wild type level. We propose that cleavage of this internal loop region plays an important regulatory function through stimulating its shedding activity. Collectively, our data provide a direct link between the pH regulation of yeast cell wall assembly and the activity of a yapsin. 相似文献
6.
H Komano N Rockwell G T Wang G A Krafft R S Fuller 《The Journal of biological chemistry》1999,274(34):24431-24437
The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation. Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg. No cleavage was seen when both Lys and Arg were absent. No significant enhancement was seen with multiple basic residues. However, cleavage always occurred carboxyl to the most COOH-terminal basic residue. V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely. These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)'. The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0. However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast. 相似文献
7.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1984,784(2-3):102-107
TWo molecular forms of casein kinase II (an ATP:protein phosphotransferase, EC 2.7.1.37) from yeast were isolated and characterized. The first form was composed of three polypeptide subunits with molecular weights of 41000, 37000 and 24000. The second form contained two larger polypeptides and lacked an autophosphorylatable 24 kDa subunit. The properties of both enzyme forms were found to be practically the same in respect to the substrate and phosphate donor specificities, kinetics, their sensitivity to heparin etc. The results obtained strongly indicate that isolated yeast casein kinase II does not necessarily require the smallest subunit for the enzyme activity. 相似文献
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Pre-elafin, also known as trappin-2, is an elastase-specific inhibitor that belongs to the trappin gene family. A chimeric gene encoding polyhistidine-tagged human pre-elafin fused to the yeast alpha-factor precursor was expressed in Saccharomyces cerevisiae. The chimera was engineered to keep a single copy of the mature alpha-factor peptide. This enabled the use of a simple bioassay (mating assay) to assess the relative efficiency of both the expression and the secretion of the recombinant molecule. We found that pre-elafin is processed both in vivo and in vitro by yapsin 1, the yeast aspartyl endoprotease encoded by YPS1. Cleavage by yapsin 1 occurred C-terminal to a subset of single lysine residues. Expression in a yapsin 1-deficient yeast strain was an indispensable condition to allow the efficient production of full-length human pre-elafin. The recombinant inhibitor was purified from concentrated culture medium by ammonium sulfate precipitation, affinity purification on a Ni(2+) resin, and cation exchange chromatography. Recombinant human pre-elafin was fully active and showed the same inhibitory profile toward different serine proteases to that reported for mature elafin. 相似文献
10.
A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm. 相似文献
11.
Nakagawa Y Sakamoto H Tateno H Hirabayashi J Oguri S 《Bioscience, biotechnology, and biochemistry》2012,76(1):25-33
A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves. 相似文献
12.
MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin
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Leroux MR Fändrich M Klunker D Siegers K Lupas AN Brown JR Schiebel E Dobson CM Hartl FU 《The EMBO journal》1999,18(23):6730-6743
Group II chaperonins in the eukaryotic and archaeal cytosol assist in protein folding independently of the GroES-like cofactors of eubacterial group I chaperonins. Recently, the eukaryotic chaperonin was shown to cooperate with the hetero-oligomeric protein complex GimC (prefoldin) in folding actin and tubulins. Here we report the characterization of the first archaeal homologue of GimC, from Methanobacterium thermoautotrophicum. MtGimC is a hexamer of 87 kDa, consisting of two alpha and four beta subunits of high alpha-helical content that are predicted to contain extended coiled coils and represent two evolutionarily conserved classes of Gim subunits. Reconstitution experiments with MtGimC suggest that two subunits of the alpha class (archaeal Gimalpha and eukaryotic Gim2 and 5) form a dimer onto which four subunits of the beta class (archaeal Gimbeta and eukaryotic Gim1, 3, 4 and 6) assemble. MtGimalpha and beta can form hetero-complexes with yeast Gim subunits and MtGimbeta partially complements yeast strains lacking Gim1 and 4. MtGimC is a molecular chaperone capable of stabilizing a range of non-native proteins and releasing them for subsequent chaperonin-assisted folding. In light of the absence of Hsp70 chaperones in many archaea, GimC may fulfil an ATP-independent, Hsp70-like function in archaeal de novo protein folding. 相似文献
13.
A multifunctional masquerade-like protein has been isolated, purified, and characterized from hemocytes of the freshwater crayfish, Pacifastacus leniusculus. It was isolated by its Escherichia coli binding property, and it binds to formaldehyde-treated Gram-negative bacteria as well as to yeast, Saccharomyces cerevisiae, whereas it does not bind to formaldehyde-fixed Gram-positive bacteria. The intact masquerade (mas)-like protein is present in crayfish hemocytes as a heterodimer composed of two subunits with molecular masses of 134 and 129 kDa. Under reducing conditions the molecular masses of the intact proteins are not changed. After binding to bacteria or yeast cell walls, the mas-like protein is processed by a proteolytic enzyme. The 134 kDa of the processed protein yields four subunits of 65, 47, 33, and 29 kDa, and the 129-kDa protein results in four subunits of 63, 47, 33, and 29 kDa in 10% SDS-PAGE under reducing conditions. The 33-kDa protein could be purified by immunoaffinity chromatography using an Ab to the C-terminal part of the mas-like protein. This subunit of the mas-like protein has cell adhesion activity, whereas the two intact proteins, 134 and 129 kDa, have binding activity to LPSs, glucans, Gram-negative bacteria, and yeast. E. coli coated with the mas-like protein were more rapidly cleared in crayfish than only E. coli, suggesting this protein is an opsonin. Therefore, the cell adhesion and opsonic activities of the mas-like protein suggest that it plays a role as an innate immune protein. 相似文献
14.
Förster C Santos MA Ruffert S Krämer R Revuelta JL 《The Journal of biological chemistry》1999,274(14):9442-9448
The vacuolar ATPase subunit A structural gene VMA1 of the biotechnologically important riboflavin overproducer Ashbya gossypii was cloned and disrupted to prevent riboflavin retention in the vacuolar compartment and to redirect the riboflavin flux into the medium. Cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequences of the Vma1 proteins from yeast and filamentous fungi. The deduced polypeptide comprises 617 amino acids with a calculated molecular mass of 67.8 kDa. The deduced amino acid sequence is highly similar to that of the catalytic subunits of Saccharomyces cerevisiae (67 kDa), Candida tropicalis (67 kDa), and Neurospora crassa (67 kDa) with 89, 87, and 60% identity, respectively, and shows about 25% identity to the beta-subunit of the FoF1-ATPase of S. cerevisiae and Schizosaccharomyces pombe. In contrast to S. cerevisiae, however, where disruption of the VMA1 gene was conditionally lethal, and to N. crassa, where viable disruptants could not be isolated, disruption of the VMA1 gene in A. gossypii did not cause a lethal phenotype. Disruption of the AgVMA1 gene led to complete excretion of riboflavin into the medium instead of retention in the vacuolar compartment, as observed in the wild type. 相似文献
15.
Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex. 总被引:2,自引:1,他引:1
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Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-kDa subunit of RPA. Rpa4 associates with the 70- and 13-kDa subunits to form a trimeric complex capable of binding to single-stranded DNA. Rpa4 is preferentially expressed in placental and colon mucosa tissues. In the placenta, Rpa4 is more abundant than the 70-kDa Rpa1 subunit and is not associated with either Rpa1 or with any other single-stranded DNA-binding protein. In proliferating cells in culture, Rpa4 is considerably less abundant than Rpa1 and Rpa2. Northern (RNA) blot analysis suggest that there are alternatively processed forms of the RPA4 mRNA, and Southern blot analysis indicates that beside RPA4 there may be other members of the RPA2 gene family. 相似文献
16.
Mammalian HDC mRNA encodes a protein with a molecular mass of 74 kDa. The reported molecular mass for the purified HDC subunit is 53-55 kDa. Western blot analysis of extracts of rat gastric mucosa and fetal rat liver has revealed the presence of at least three different forms of HDC immunoreactivity, having molecular masses of about 74, 63 and 53 kDa. There is evidence from previous studies that full length rat HDC is enzymatically inactive and that activation requires C-terminal truncation. In the present study we examined the various immunoreactive HDC forms in rat oxyntic mucosa and their response to treatments known to affect the HDC activity. Freely fed rats and hypergastrinemic rats (treated with gastrin or the proton pump inhibitor omeprazole) had higher oxyntic mucosal HDC activity and HDC mRNA level than fasted or untreated rats. The difference in HDC activity was greater than the difference in HDC mRNA level. Western blot analysis confirmed the existence of the 74, 63 and 53 kDa HDC forms in the oxyntic mucosa. All three forms were more abundant in the oxyntic mucosa of freely fed and hypergastrinemic rats than in the mucosa of fasted or untreated rats. Of the three HDC forms, the 63 kDa form was the predominant one, the 73 kDa form was quantitatively insignificant by comparison and the 53 kDa form was at or below the limit of detection in fasted rats. The activity of HDC was well correlated to the amount of the 63 kDa HDC form. Administration of cycloheximide to hypergastrinemic rats (undergoing omeprazole treatment) resulted in a rapid decline of the HDC activity (estimated half-life 1 h and 50 min). The 63 kDa HDC form disappeared with a rate that corresponded to the decline in HDC activity. The two other HDC forms seemed to have a slower turnover. Our findings suggest that the 63 kDa form is enzymatically active. The results do not allow any conclusion as to the functional activity of the 74 and 53 kDa forms. 相似文献
17.
Carboxylester lipase (cholesterol esterase, EC 3.1.1.13) has been purified to homogeneity from porcine pancreas. The enzyme is isolated in two molecular mass forms, a monomer of 74 kDa and a dimer of 167 kDa. The dimer consists of two catalytically-active subunits which have molecular masses approximately 9 kDa greater than the monomers. The difference in size was not attributable to carbohydrate or lipid content. The catalytic properties of the two forms are comparable on a weight basis, the amino acid compositions are quite similar, and the N-terminal sequences are nearly identical for 24 residues. These similarities suggest a possible precursor-product relationship between the two carboxylester lipase forms. 相似文献
18.
The synthesis, accumulation, and cellular distribution of cathepsins E and D during the dimethyl sulfoxide (DMSO)-induced differentiation of Friend erythroleukemia cells were investigated. The cellular levels of cathepsins E and D rapidly increased within 1 day of DMSO induction and then sharply decreased over the next 7 days. Since the cells during 1 day of differentiation were morphologically the same as uninduced cells, the results suggest the importance of these enzymes in more cellular proteolysis for the following committed differentiation. While cathepsin D was present mostly in the sedimentable fraction of cells throughout the differentiation period, the distribution of cathepsin E varied to the stage of differentiation. The ratio of the soluble/sedimentable cathepsin E content was 1.1, 1.4, 0.9, and 0.7 in cells after 0, 1, 4, and 7 days of DMSO treatment, respectively. The maturation of reticulocytes to erythrocytes was accompanied by complete loss of the soluble cathepsin E and of all of the cellular cathepsin D. Immunoblotting analyses revealed that both uninduced and induced cells contained two forms of cathepsin E; a high molecular weight form (82 kDa) which was mainly associated with the sedimentable fraction and a low molecular weight form (74 kDa) which was found largely in the soluble fraction. The distribution of these two forms was not significantly changed throughout the differentiation period, but the 74-kDa protein was completely eliminated with maturation of reticulocytes to erythrocytes. Cathepsin D also appeared in two forms in both uninduced and induced cells; a minor (46 kDa) and a major (42 kDa) form which appear to have a precursor-product relationship. 相似文献
19.
Egel-Mitani M Andersen AS Diers I Hach M Thim L Hastrup S Vad K 《Enzyme and microbial technology》2000,26(9-10):671-677
Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible. 相似文献
20.
Soluble carnitine acetyltransferase from Candida tropicalis is synthesized as a 76 kDa precursor, which is monomeric and possesses no or very little carnitine acetyltransferase activity. Maturation of the enzyme begins with proteolytic processing of the 76 kDa precursor to 64 and 57 kDa subunits. The processed subunits subsequently associate into two kinds of active oligomers; the 57 kDa subunits are assembled into a tetramer and the 64 kDa subunits into an octamer. Formation of these oligomers depends apparently on growth conditions, since both oligomers were present in cells grown in continuous culture, but cells grown batchwise contained only the tetrameric form of carnitine acetyltransferase. 相似文献