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1.
Ma Z  Csuhai E  Chow KM  Hersh LB 《Biochemistry》2001,40(31):9447-9452
Kinetic evidence suggests an acidic region in nardilysin binds polyamines and acts as a regulatory domain. The binding of approximately 5 mol of spermine/mol of nardilysin was demonstrated. The binding curve was sigmoidal exhibiting an IC(50) of approximately 118 microM and a Hill coefficient of 1.8. Spermine diminished the tryptophan fluorescence of the enzyme and increased its sensitivity to protease V8. The acidic stretch from mouse and human nardilysin were expressed as glutathione transferase fusion proteins. All fusion proteins bound spermine with an IC(50) of 40 to 110 microM. The mouse fusion protein bound approximately 7 mol of spermine exhibiting a sigmoidal binding curve and a Hill coefficient of 1.4. The human acidic stretch, containing fewer acidic residues, bound approximately 5 mol of spermine/mol with a hyperbolic binding curve. Chimeric fusion proteins containing the N-terminus of the mouse acidic region fused to the C-terminus of the human acidic region bound approximately 10 mol of spermine, while the opposite chimera bound approximately 4 mol of spermine/mol. The N-terminal region of the mouse acidic domain binds 3--4 mol spermine/mol exhibiting a Hill coefficient of 1.4, while the same region from human nardilysin binds 1 mol of spermine/mol. Spermine enhanced the sensitivity of the mouse acidic domain, but not the human acidic domain, to protease V8. Together the data support a model where the acidic stretch of nardilysin functions as an autonomous domain.  相似文献   

2.
Catalase (hydroperoxidase) HPII of Escherichia coli is the largest catalase so far characterized, existing as a homotetramer of 84 kDa subunits. Each subunit has a core structure that closely resembles small subunit catalases, supplemented with an extended N-terminal sequence and compact flavodoxin-like C-terminal domain. Treatment of HPII with trypsin, chymotrypsin, or proteinase K, under conditions of limited digestion, resulted in cleavage of 72-74 residues from the N-terminus of each subunit that created a homotetramer of 76 kDa subunits with 80% of wild-type activity. Longer treatment with proteinase K removed the C-terminal domain, producing a transient 59 kDa subunit which was subsequently cleaved into two fragments, 26 and 32 kDa. The tetrameric structure was retained despite this fragmentation, with four intermediates being observed between the 336 kDa native form and the 236 kDa fully truncated form corresponding to tetramers with a decreasing complement of C-termini (4, 3, 2, and 1). The truncated tetramers retained 80% of wild-type activity. The T(m) for loss of activity during heating was decreased from 85 to 77 degrees C by removal of the N-terminal sequence and to 59 degrees C by removal of the C-terminal domain, revealing the importance of the C-terminal domain in enzyme stability. The sites of cleavage were determined by N- and C-terminal sequencing, and two were located on the surface of the tetramer with a third being exposed by removal of the C-terminal domain.  相似文献   

3.
Methionyl-tRNA synthetase (MetRS, 2 X 75 kDa) was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB8. The polypeptide chain of MetRS was cleaved by limited digestion with trypsin into four domains: T1 (29 kDa), T2 (23 kDa), T3 (14.5 kDa), and T4 (7.5 kDa), which were aligned in that order. MetRS was also cleaved into similar fragments with a variety of other proteases. Domains T1, T2, T3, and T4 were isolated by column chromatography. "Tandem domain" T1-T2 (56 kDa) is fully active in the aminoacylation of tRNA and is further cleaved with trypsin into domains T1 and T2. Domain T1 is the smallest aminoacylation unit so far reported. Domain T2 (enzymatically inactive) interacts with tRNAMetf, as found by UV-induced cross-linking. Isolated domain T3 forms a dimer and is responsible for the dimer assembly of two protomers in MetRS. Domain T4 is a flexible tail of MetRS. These domains, in particular T1 and T2, will be important for detailed structure analyses in relation to aminoacylation activity.  相似文献   

4.
Gel filtration chromatography showed that nardilysin activity in a rat testis or rat brain extract exhibited an apparent molecular weight of approximately 300 kDa compared to approximately 187 kDa for the purified enzyme. The addition of purified nardilysin to a rat brain extract, but not to an E. coli extract, produced the higher molecular species. The addition of a GST fusion protein containing the acidic domain of nardilysin eliminated the higher molecular weight nardilysin forms, suggesting that oligomerization involves the acidic domain of nardilysin. Using an immobilized nardilysin column, mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) were isolated from a fractionated rat brain extract. Porcine mMDH, but not porcine cytosolic MDH, was shown to form a heterodimer with nardilysin. Mitochondrial MDH increased nardilysin activity about 50%, while nardilysin stabilized mMDH towards heat inactivation. CS was co-immunoprecipitated with mMDH only in the presence of nardilysin showing that nardilysin facilitates complex formation.  相似文献   

5.
Vip3 is a novel insect toxin isolated from Bacillus thuringiensis (Bt), and could be used as an alternative toxin for Bt δ-endotoxins for transgenic insect control. Vip3 mutants with deletion, addition or mutations at the very end of the C-terminus were generated. The deletion and addition of a few amino acid residues at the C-terminus totally abolished the insecticidal activity. The mutation of the last two residues from IK to LG also resulted in the total loss of its insecticidal activity; however, the mutation from IK to LR increased its activity substantially against beet armyworm. Interestingly, all the inactive mutants were found to be highly sensitive to trypsin digestion, while the active mutant generated a trypsin-resistant polypeptide of 62-kDa upon trypsin digestion. However, this 62-kDa polypeptide expressed in E. coli from the 5' end truncated vip3 gene was biologically inactive and sensitive to trypsin digestion. Thus, the N-terminal part of the protein is required to form the 62-kDa trypsin resistant core. This study suggested that the 62-kDa peptide core could be a hallmark of active insecticidal Vip3 proteins.  相似文献   

6.
7.
H J Goren  M F White  C R Kahn 《Biochemistry》1987,26(8):2374-2382
We have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the beta-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22 degrees C with low concentrations (5-10 micrograms/mL, pH 7.4) of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the beta-subunit was carried out before and after digestion, and the [32P]phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and analyzed by tryptic peptide mapping by use of reverse-phase high-performance liquid chromatography. Mild trypsin digestion reduced the apparent molecular mass of the beta-subunit from 95 to 85 kDa, and then to 70 kDa. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the beta-subunit (alpha Pep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the [32P]phosphate originally found in the beta-subunit, and tryptic peptide mapping showed that two major tryptic phosphopeptides (previously called pY2 and pY3) were removed. Three other tryptic phosphopeptides (pY1, pY1a, and pY4) were found in the 85- and 70-kDa fragments. Treatment of the intact receptor with staphylococcal V8 protease also converted the beta-subunit to an 85-kDa fragment that did not bind to alpha Pep-1, contained about 50% of the initial radioactivity, and lacked pY2 and pY3. Elastase rapidly degraded the receptor to inactive fragments between 37 and 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.  相似文献   

9.
10.
Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.  相似文献   

11.
In the absence of cAMP the cyclic AMP receptor protein (CRP) is relatively resistant to trypsin whereas the cAMP X CRP complex is attacked yielding N-terminal core fragments of 14,300 and 18,500 Da which still bind cAMP. The DNA X CRP complex formed at low ionic strength in the absence of cAMP is cleaved by trypsin with the formation of 9,700- and 6,000-Da fragments and the concomitant loss of cAMP binding activity. DNA X CRP remains as resistant to attack by subtilisin, clostripain, and the Staphylococcus aureus V8 protease as unliganded CRP but is slowly digested by chymotrypsin. All of the double-stranded polydeoxyribonucleotides and several of the single-stranded polydeoxyribonucleotides and polyribonucleotides tested render CRP sensitive to cleavage by trypsin. CRP is less rapidly cleaved by trypsin in the presence of d(A)n, d(I)n, and r(C)n indicative of a weaker affinity of CRP for these polynucleotides. The 9,700-Da fragment is N-terminal in CRP and probably terminates at Lys-89. The loss of cAMP binding activity following trypsin cleavage of DNA X CRP indicates that regions beyond this residue are important in the function of the cAMP-binding domain of CRP. The 6,000-Da fragment extends from Val-131 to Arg-185 or Lys-188 and contains part of the F helix involved in DNA binding by CRP.  相似文献   

12.
The thermal unfolding of myosin subfragment 1 (S1) cleaved by trypsin was studied by differential scanning calorimetry. In the absence of nucleotides, trypsin splits the S1 heavy chain into three fragments (25, 50, and 20 kDa). This cleavage has no appreciable influence on the thermal unfolding of S1 examined in the presence of ADP, in the ternary complexes of S1 with ADP and phosphate analogs, such as orthovanadate (Vi) or beryllium fluoride (BeFx), and in the presence of F-actin. In the presence of ATP and in the complexes S1.ADP.Vi or S1.ADP.BeFx, trypsin produces two additional cleavages in the S1 heavy chain: a faster cleavage in the N-terminal region between Arg23 and Ile24, and a slower cleavage at the 50 kDa fragment. It has been shown that the N-terminal cleavage strongly decreases the thermal stability of S1 by shifting the maximum of its thermal transition by about 7 degrees C to a lower temperature, from 50 degrees C to 42.4 degrees C, whereas the cleavage at both these sites causes dramatic destabilization of the S1 molecule leading to total loss of its thermal transition. Our results show that S1 with ATP-induced N-terminal cleavage is able, like uncleaved S1, to undergo global structural changes in forming the stable ternary complexes with ADP and Pi analogs (Vi, BeFx). These changes are reflected in a pronounced increase of S1 thermal stability. However, S1 cleaved by trypsin in the N-terminal region is unable, unlike S1, to undergo structural changes induced by interaction with F-actin that are expressed in a 4-5 degrees C shift of the S1 thermal transition to higher temperature. Thus, the cleavage between Arg23 and Ile24 does not significantly affect nucleotide-induced structural changes in the S1, but it prevents structural changes that occur when S1 is bound to F-actin. The results suggest that the N-terminal region of the S1 heavy chain plays an important role in structural stabilization of the entire motor domain of the myosin head, and a long-distance communication pathway may exist between this region and the actin-binding sites.  相似文献   

13.
We investigated the effect of divalent metal ions on the proteolytic cleavage and activation of platelet Factor XIII by thrombin and trypsin. In the absence of metal ions (5 mM EDTA), trypsin and thrombin rapidly degraded platelet Factor XIII (80 kDa) to low-molecular-mass peptides (50-19 kDa) with simultaneous loss of transglutaminase activity. Divalent metal ions protected Factor XIII from proteolytic inactivation with an order of efficacy of Ca2+ greater than Zn2+ greater than Mg2+ greater than Mn2+. Calcium (2 mM) increased by 10- to 1000-fold the trypsin and thrombin concentrations required to degrade Factor XIII to a 19-kDa peptide. Factor XIIIa formed by thrombin in the presence of 5 mM EDTA had one-half the specific activity of Factor XIIIa formed in the presence of calcium. Factor XIII was cleaved by trypsin in the presence of 5 mM Ca2+ to a 51 +/- 3-kDa fragment that had 60% of the original Factor XIIIa activity. A similar tryptic peptide formed in the presence of 5 mM EDTA did not have transglutaminase activity. In the presence of 5 mM Mg2+, thrombin cleaved Factor XIII to a major 51 +/- 3-kDa fragment that had 60% of the Factor XIIIa activity. Mn2+ (0.1-5 mM) limited trypsin and thrombin proteolysis. The resulting digest containing a population of Factor XIII fragments (50-14 kDa) expressed 50-60% transglutaminase activity of Factor XIIIa. Factor XIII was fully activated by both trypsin and thrombin in the presence of 5 mM Zn2+, resulting in two fragments of 76 and 72 kDa. We conclude that the binding of divalent metal ions to platelet Factor XIII induces conformational changes in the protein that alter its susceptibility to proteolysis and influence the expression of transglutaminase activity.  相似文献   

14.
The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.  相似文献   

15.
Botulinum neurotoxin type E, a 150 kDa single chain protein, cleaved with endoproteinase Lys-C yielded 113, 73, and 50 kDa fragments. The N-terminal sequence of the 113 kDa fragment, Gly-Ile-Arg-Lys-Ser-Ile-Cys-Ile, overlaps the N-terminal sequence, Lys-Ser-Ile-Cys-Ile, of the 103 kDa heavy chain produced by nicking the neurotoxin with trypsin. The -Arg-Lys- bond is therefore the site on the single chain type E NT where trypsin nicks generating the 50 kDa light and 103 kDa heavy chains of the dichain NT. The sequence of the first 50 N-terminal residues of the 73 kDa fragment were determined. This fragment is a segment of the heavy chain; 50% of the 50 residues are present in identical positions in a similar segment of the heavy chain of tetanus neurotoxin.  相似文献   

16.
The storage tissues of many plants contain protease inhibitors that are believed to play an important role in defending the plant from invasion by pests and pathogens. These proteinaceous inhibitor molecules belong to a number of structurally distinct families. We describe here the isolation, purification, initial inhibitory properties, and three-dimensional structure of a novel trypsin inhibitor from seeds of Veronica hederifolia (VhTI). The VhTI peptide inhibits trypsin with a submicromolar apparent K(i) and is expected to be specific for trypsin-like serine proteases. VhTI differs dramatically in structure from all previously described families of trypsin inhibitors, consisting of a helix-turn-helix motif, with the two alpha helices tightly associated by two disulfide bonds. Unusually, the crystallized complex is in the form of a stabilized acyl-enzyme intermediate with the scissile bond of the VhTI inhibitor cleaved and the resulting N-terminal portion of the inhibitor remaining attached to the trypsin catalytic serine 195 by an ester bond. A synthetic, truncated version of the VhTI peptide has also been produced and co-crystallized with trypsin but, surprisingly, is seen to be uncleaved and consequently forms a noncovalent complex with trypsin. The VhTI peptide shows that effective enzyme inhibitors can be constructed from simple helical motifs and provides a new scaffold on which to base the design of novel serine protease inhibitors.  相似文献   

17.
The MET proto-oncogene encodes a 190-kDa disulfide-linked heterodimeric receptor (p190 alpha beta) whose tyrosine kinase activity is triggered by the hepatocyte growth factor. The mature receptor is made of two subunits: an alpha chain of 50 kDa and a beta chain of 145 kDa, arising from proteolytic cleavage of a single-chain precursor of 170 kDa (pr170). In a colon carcinoma cell line (LoVo), the precursor is not cleaved and the Met protein is exposed at the cell surface as a single-chain polypeptide of 190 kDa (p190NC). The expression of the uncleaved Met protein is due to defective posttranslational processing, since in this cell line (i) the proteolytic cleavage site Lys-303-Arg-Lys-Lys-Arg-Ser-308 is present in the precursor, (ii) p190NC is sensitive to mild trypsin digestion of the cell surface, generating alpha and beta chains of the correct size, and (iii) the 205-kDa insulin receptor precursor is not cleaved as well. p190NC is a functional tyrosine kinase in vitro and is activated in vivo, as shown by constitutive autophosphorylation on tyrosine. The MET gene is neither amplified nor rearranged in LoVo cells. Overlapping cDNA clones selected from a library derived from LoVo mRNA were sequenced. No mutations were present in the MET-coding region. These data indicate that the tyrosine kinase encoded by the MET proto-oncogene can be activated as a consequence of a posttranslational defect.  相似文献   

18.
The mature human immunodeficiency virus type 1 proteinase (PR; 11 kDa) can cleave all interdomain junctions in the Gag and Gag-Pol polyprotein precursors. To determine the activity of the enzyme in its precursor form, we blocked release of mature PR from a truncated Gag-Pol polyprotein by introducing mutations into the N-terminal Phe-Pro cleavage site of the PR domain. The mutant precursor autoprocessed efficiently upon expression in Escherichia coli. No detectable mature PR was released; however, several PR-related products ranging in size from approximately 14 to 18 kDa accumulated. Products of the same size were generated when mutant precursors were digested with wild-type PR. Thus, PR can utilize cleavage sites in the region upstream of the PR domain, resulting in the formation of extended PR species. On the basis of active-site titration, the PR species generated from mutated precursor exhibited wild-type activity on peptide substrates. However, the proteolytic activity of these extended enzymes on polyprotein substrates provided exogenously was low when equimolar amounts of extended and wild-type PR proteins were compared. Mammalian cells expressing the mutated precursor produced predominantly precursor and considerably reduced amounts of mature products. Released particles consisted mostly of uncleaved or partially cleaved polyproteins. Our results suggest that precursor forms of PR can autoprocess but are less efficient in processing of the Gag precursor for formation of mature virus particles.  相似文献   

19.
Cytochrome c and dATP/ATP induce oligomerization of Apaf-1 into two distinct apoptosome complexes: an approximately 700 kDa complex, which recruits and activates caspases-9, -3 and -7, and an approximately 1.4 MDa complex, which recruits and processes caspase-9, but does not efficiently activate effector caspases. While searching for potential inhibitors of the approximately 1.4 MDa apoptosome complex, we observed an approximately 30 kDa Apaf-1 immunoreactive fragment that was associated exclusively with the inactive complex. We subsequently determined that caspase-3 cleaved Apaf-1 within its CED-4 domain (SVTD(271) downward arrowS) in both dATP-activated lysates and apoptotic cells to form a prominent approximately 30 kDa (p30) N-terminal fragment. Purified recombinant Apaf-1 p30 fragment weakly inhibited dATP-dependent activation of caspase-3 in vitro. However, more importantly, prevention of endogenous formation of the p30 fragment did not stimulate latent effector caspase processing activity in the large complex. Similarly, the possibility that XIAP, an inhibitor of apoptosis protein (IAP), was responsible for the inactivity of the approximately 1.4 MDa complex was excluded as immunodepletion of this caspase inhibitor failed to relieve the inhibition. However, selective proteolytic digestion of the approximately 1.4 MDa and approximately 700 kDa complexes showed that Apaf-1 was present in conformationally distinct forms in these two complexes. Therefore, the inability of the approximately 1.4 MDa apoptosome complex to process effector caspases most likely results from inappropriately folded or oligomerized Apaf-1.  相似文献   

20.
H C Palfrey 《FEBS letters》1983,157(1):183-190
Incubation of cytosol fractions from a variety of mammalian tissues (heart, liver, lung, adrenal, spleen and skeletal muscle) with Ca2+ (0.5 mM) in the presence of gamma-[32P]ATP resulted in the phosphorylation of a prominent substrate of Mr approximately 100 000 (100 kDa). One-dimensional peptide maps and two-dimensional tryptic fingerprints of the phosphoprotein from these sources were identical. A single major phosphopeptide was generated by trypsin and was determined to contain exclusively phosphothreonine. The 100 kDa substrate could be distinguished from glycogen phosphorylase (Mr approximately 97 000) by a number of criteria including phosphopeptide mapping and by its failure to bind either to glycogen or to a specific antiphosphorylase antibody. The Ca2+-dependent protein kinase responsible for phosphorylation of the 100 kDa protein appeared to be a calmodulin (CaM)-requiring enzyme in that it could be inhibited in cytosol extracts by trifluoperazine (IC50 6-16 microM) and that exogenous CaM was necessary for 100 kDa phosphorylation in CaM-depleted cytosol. These results suggest that a rise in intracellular Ca2+ resulting in an activation of CaM-dependent protein kinase leads to the phosphorylation of a common 100 kDa substrate in many tissues.  相似文献   

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