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1.
Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase 总被引:8,自引:0,他引:8
S A Kuby R H Palmieri A Frischat A H Fischer L H Wu L Maland M Manship 《Biochemistry》1984,23(11):2393-2399
The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
I Kay M Patel G M Coast N F Totty A I Mallet G J Goldsworthy 《Regulatory peptides》1992,42(3):111-122
A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides. 相似文献
3.
In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model 总被引:1,自引:0,他引:1
H J Kim S Nishikawa Y Tokutomi H Takenaka M Hamada S A Kuby S Uesugi 《Biochemistry》1990,29(5):1107-1111
Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site. 相似文献
4.
Amino acid changes in the Sindbis virus E2 glycoprotein that increase neurovirulence improve entry into neuroblastoma cells. 总被引:1,自引:1,他引:0
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Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and results in age-dependent mortality. The outcome is dependent on the virus strain. Residues at 55 and 172 in the E2 glycoprotein determine the neurovirulence for mice of different ages and the efficiency of replication in the nervous system and neuronal cells. To determine the effects of these two residues on the initial steps in replication, we studied viruses with a histidine or glutamine at E2 position 55 and a glycine or an arginine at position 172, E2[H55G172], E2[Q55G172], E2[H55R172], and E2[Q55R172]. The production of virus was detected earlier for viruses with a histidine at E2 position 55 in BHK-21 cells (4 to 6 versus 6 to 8 h) and for E2[H55G172] in N18 cells (6 versus 8 to 10 h). As shown previously, viruses with a glycine at E2 position 172 bound more efficiently to N18 cells and a histidine at E2 position 55 further improved binding only slightly. Viruses with E2[H55] exhibited more rapid internalization and degradation of viral proteins in both BHK-21 and N18 cells. Incubation of E2[H55G172] and E2[Q55G172] at various pHs and temperatures did not reveal differences in virion stability. These data suggest that the amino acids at E2 positions 172 and 55 affect both adsorption and penetration of SV and that these early steps in the replicative pathway contribute to increased neurovirulence. 相似文献
5.
6-Hydroxybenzofuran and phenylhydrazine are mechanism-based inhibitors of dopamine beta-hydroxylase (D beta H; EC 1.14.17.1). We report here the isolation and characterization of radiolabeled peptides obtained after inactivation of D beta H with [3H]6-hydroxybenzofuran and [14C]phenylhydrazine followed by digestion with Staphylococcus aureus V8 protease. Inactivation of D beta H with [3H]6-hydroxybenzofuran gave only one labeled peptide, whereas inactivation with [14C]phenylhydrazine gave several labeled peptides. Each inhibitor labeled a unique tyrosine in the enzyme corresponding to Tyr477 in the primary sequence of the bovine enzyme (Robertson, J. G., Desai, P. R., Kumar, A., Farrington, G. K., Fitzpatrick, P. F., and Villafranca, J. J. (1990) J. Biol. Chem. 265, 1029-1035). In addition, [14C]phenylhydrazine also labeled a unique histidine (His249) as well as several other peptides. Examination of the complete peptide profile obtained by high pressure liquid chromatography analysis also revealed the presence of a modified but nonradioactive peptide. This peptide was isolated and sequenced and was identical whether the enzyme was inactivated by 6-hydroxybenzofuran or phenylhydrazine. An arginine at position 503 was missing from the sequence cycle performed by Edman degradation of the modified peptide, but arginine was present in the identical peptide isolated from native dopamine beta-hydroxylase. These data are analyzed based on an inactivation mechanism involving formation of enzyme bound radicals (Fitzpatrick, P. F., and Villafranca, J. J. (1986) J. Biol. Chem. 261, 4510-4518) interacting with active site amino acids that may have a role in substrate binding and binding of the copper ions at the active site. 相似文献
6.
M Montemartini H M Kalisz H J Hecht P Steinert L Flohé 《European journal of biochemistry》1999,264(2):516-524
Tryparedoxin peroxidase (TXNPx), recently identified as the hydroperoxide-detoxifying enzyme of trypanosomatidae [Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M. & Flohé, L. (1997) Biol. Chem. 378, 827-836], is a member of the peroxiredoxin family and is characterized by two VCP motifs. Based on a consensus sequence of TXNPx and peroxiredoxin-type peroxidases, eight TXNPx variants were designed, heterologously expressed in Escherichia coli, checked for alpha-helix content by CD and kinetically analysed. The variant Q164E was fully active, C52S, W87D and R128E were inactive and C173S, W87H, W177E and W177H showed reduced activity. Wild-type TXNPx and Q164E exhibit ping-pong kinetics with infinite maximum velocities, whereas saturation kinetics were observed with C173S and W177E. The data comply with a mechanism in which C52, primarily activated by R128 and possibly by W87, is first oxidized by hydroperoxide to a sulfenic acid derivative. C173, supported by W177, then forms an intersubunit disulfide bridge with C52. If C173 is exchanged with a redox-inactive residue (Ser) or is insufficiently activated, the redox shuttle remains restricted to C52. The shift in the kinetic pattern and decrease in specific activity of C173S and W177E may result from a limited accessibility of the oxidized C52 to tryparedoxin, which in the oxidized wild-type TXNPx presumably attacks the C173 sulfur of the disulfide bridge. The proposed mechanism of action of TXNPx is consistent with that deduced for the homologous thioredoxin peroxidase of yeast [Chae, H. Z., Uhm, T. B. & Rhee, S. G. (1994) Proc. Natl Acad. Sci. USA 91, 7022-7026] and is supported by molecular modelling based on the structure of the human peroxiredoxin 'hORF6' [Choi, H.-J., Kang, S. W. Yang, C.-H., Rhee, S. G. & Ryu, S.-E. (1998) Nat. Struct. Biol. 5, 400-406]. 相似文献
7.
8.
The primary structure of skeletal muscle myosin heavy chain: IV. Sequence of the rod, and the complete 1,938-residue sequence of the heavy chain 总被引:6,自引:0,他引:6
T Maita E Yajima S Nagata T Miyanishi S Nakayama G Matsuda 《Journal of biochemistry》1991,110(1):75-87
In the preceding paper [Maita, T., Miyanishi, T., Matsuzono, K., Tanioka, Y., & Matsuda, G. (1991) J. Biochem. 110, 68-74], we reported the amino-terminal 837-residue sequence of the heavy chain of adult chicken pectoralis muscle myosin. This paper describes the carboxyl terminal 1,097-residue sequence and the linkage of the two sequences. Rod obtained by digesting myosin filaments with alpha-chymotrypsin was redigested with the protease at high KCl concentration, and two fragments, subfragment-2 and light meromyosin, were isolated and sequenced by conventional methods. The linkage of the two fragments was deduced from the sequence of an overlapping peptide obtained by cleaving the rod with cyanogen bromide. The rod contained 1,039 amino acid residues, but lacked the carboxyl-terminal 58 residues of the heavy chain. A carboxyl-terminal 63-residue peptide obtained by cleaving the whole heavy chain with cyanogen bromide was sequenced. Thus, the carboxyl terminal 1,097-residue sequence of the heavy chain was completed. The linkage of subfragment-1 and the rod was deduced from the sequence of an overlapping peptide between the two which was obtained by cleaving heavy meromyosin with cyanogen bromide. Comparing the sequence of the adult myosin thus determined with that of chicken embryonic myosin reported by Molina et al. [Molina, M.I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488], we found that the sequence homology is 94%. 相似文献
9.
An alamethicin, secreted by the fungus Trichoderma viride and containing a glutamine at position 18 instead of the usual glutamic acid, has been uniformly labeled with 15N and purified by HPLC. The extent of 15N incorporation at individual backbone and side-chain sites was found to vary from 85% to 92%, as measured by spin-echo difference spectroscopy. The proton NMR spectrum of the peptide dissolved in methanol was assigned using correlation spectroscopies and nuclear Overhauser enhancements (NOE) measured in the rotating frame. The 15N resonances were assigned by the 2D 1H-15N correlation via heteronuclear multiple-quantum coherence experiment. NOEs and 3JNHC alpha H coupling constants strongly suggest that, in methanol, from Aib-3 to Gly-11, the peptide adopts a predominantly helical conformation, in agreement with previous 1H NMR studies [Esposito, G., Carver, J.A, Boyd, J., & Campbell, I.D. (1987) Biochemistry 26, 1043-1050; Banerjee, U., Tsui, F.-P., Balasubramanian, T.N., Marshall, G.R., & Chan, S I. (1983) J. Mol. Biol. 165, 757-775]. The conformation of the carboxyl terminus (12-20) is less well determined, partly because the amino acid composition reduces the number of NOEs and coupling constants which can be determined by 1H NMR spectroscopy. The 3JNHC alpha H in the C-terminus suggest the possibility of conformational averaging at Leu-12, Val-15, and Gln-19, an interpretation which is supported by a recent molecular dynamics simulation of the peptide [Fraternalli, F. (1990) Biopolymers 30, 1083-1099].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
Tissue plasminogen activator, separated into variants I and II (differing in Mr by 2000-3000), was reduced and [14C]carboxymethylated. Fragments from cleavages with enzymes and cyanogen bromide (CNBr) were separated by reverse-phase high-performance liquid chromatography and subjected to sequence degradations. All seven CNBr fragments were purified and found to be compatible with the cDNA-derived amino acid sequence [Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Ylverton, E., Seeburg, P. H., Heynecker, H. L., Goeddel, D. V., & Collen, D. (1983) Nature (London) 301, 214-221]. Chemical characterization of 93% of the 527 residues recovered in 50 peptides confirmed the indirectly deduced primary structure of the protein. The tryptic peptide patterns from the two variants were found to differ for one peptide (T15). Since carbohydrate was present in this peptide for variant I and since a marked difference in chromatographic behavior for T15 was observed in variant II, we conclude that carbohydrate differences in this peptide (i.e., Asn-184 in the numbering system of the cDNA-derived amino acid sequence) are the explanation for the size differences between variants I and II. Carbohydrate was also found at two other positions in the protein, corresponding to Asn-117 and Asn-448. However, a fourth potential glycosylation site, Asn-218, is apparently not utilized for carbohydrate attachment. The enzyme is inactivated by diisopropyl phosphorofluoridate, which covalently modifies the serine residue corresponding to position 478, identifying this as the active site serine residue.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor 总被引:23,自引:0,他引:23
A Marmenout L Fransen J Tavernier J Van der Heyden R Tizard E Kawashima A Shaw M J Johnson D Semon R Müller 《European journal of biochemistry》1985,152(3):515-522
U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region. 相似文献
12.
The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda 总被引:2,自引:0,他引:2
The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed. 相似文献
13.
The flaccid paralysis in the neuromuscular disease botulism appears to depend on the coordinated roles of the approximately 50 kDa light and approximately 100 kDa heavy chain subunits of the approximately 150 kDa neurotoxic protein produced by Clostridium botulinum (J. Biol. Chem. (1987) 262, 2660 and Eur. J. Biochem. (1988) 177, 683). We observed that the light chain after separation from its conjugate heavy chain, in the presence of dithiothreitol and 2 M urea, begins to split into approximately 28 and approximately 18 kDa fragments. The other subunit-the approximately 100 kDa heavy chain following its isolation-and the parent approximately 150 kDa dichain neurotoxin do not break down under comparable conditions. This cleavage was examined in the neurotoxin serotypes A and E. The cleavage does not appear to be due to a protease. Partial amino acid sequences established that: i) the approximately 28-kDa and approximately 18-kDa fragments comprise the N- and C-terminal regions of the light chain, respectively; ii) the light chain of the neurotoxin serotypes A and E break down at precise peptide bonds; iii) the peptide bonds cleaved in serotypes A and E are five residues apart; and iv) the portions of the approximately 18 kDa fragments of serotype A and E neurotoxin sequenced so far are highly homologous to the corresponding region of tetanus neurotoxin produced by Clostridium tetani. The partial N-terminal sequence of the approximately 28 kDa fragment matches with the N-terminal sequence of the intact L chain. The 47 residues of the approximately 18-kDa fragment of type A sequenced from its N-terminal are: -Y.E.M.S.G.L.E.V.S.F.E.E.L.R.T.F.G.G.H.D.A.K.F.I.D.S.L.Q.E.N.E.F.R.L.Y.Y .Y. N.K.F.K. D.I.A.S.T.L.-. These align with those of tetanus neurotoxin beginning at its residue #259 (Tyr); the 18 underlined residues of the above 47 residues (i.e. 38%) are identical in positions between the two proteins. The 41 residues sequenced from the approximately 18 kDa fragment of type E botulinum neurotoxin are: -K.G.I.N.I.E.E.F.L. T.F.G.N.N.D.L.N.I.I.T.V.A.Q.Y.N.D.I.Y.T.N.L.L.N.D.Y.R. K.I.A.X.K. L.-.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
J C Lagarias A V Klotz J L Dallas A N Glazer J E Bishop J F O'Connell H Rapoport 《The Journal of biological chemistry》1988,263(26):12977-12985
Previous spectroscopic studies on the phycocyanobilin-containing peptide beta-2T from Synechococcus sp. 6301 C-phycocyanin and the phycoerythrobilin-containing peptide beta-2TP from Porphyridium cruentum B-phycoerythrin indicated a different single thioether mode of attachment, postulated to be through the D-ring of the tetrapyrrole, in contrast to the A-ring linkage established for the other singly linked bilins in these proteins (Bishop, J.E., Lagarias, J.C., Nagy, J. O., Schoenleber, R.W., Rapoport, H., Klotz, A.V., and Glazer, A.N. (1986) J. Biol. Chem. 261, 6790-6796; Klotz, A.V., Glazer, A.N., Bishop, J.E., Nagy, J.O., and Rapoport, H. (1986) J. Biol. Chem. 261, 6797-6805). The crystal structure of Agmenellum quadruplicatum C-phycocyanin at 2.5-A resolution (Schirmer, T., Bode, W., and Huber, R. (1987) J. Mol. Biol., 196, 677-695) supports an A-ring linkage for all three phycocyanobilins. Consequently we have re-evaluated our proposed structural assignments by further 1H NMR studies. Two-dimensional homonuclear correlated and nuclear Overhauser enhancement spectroscopic data presented here show that all three bilins in Synechococcus 6301 C-phycocyanin are attached solely through the A-ring, complementary to the crystallographic data. The evidence from the NMR data for all bilin peptides examined includes the dipoledipole interactions of the 5-H with the 3-H, 3'-H, and a pyrrole methyl group (7-CH3); the corresponding interactions would not be possible in a D-ring-linked bilin. The 5-H also consistently exhibits allylic J-coupling to the 3-H, supporting A-ring linkage assignment. These data are inconsistent with the alternative D-ring linkage assignment since this would involve J-coupling through five bonds. Examination of the phycoerythrobilin beta-2 position in B-phycoerythrin also reveals an A-ring type of attachment by similar criteria. We conclude that all singly linked bilins are attached through the A-ring. 相似文献
15.
The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptides from digestion with a staphylococcal proteinase. 总被引:5,自引:4,他引:1
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The extracellular proteinase of Staphylococcus aureus strain V8 was used to digest the NADP-specific glutamate dehydrogenase of Neurospora crassa. Of 35 non-overlapping peptides expected from the glutamate content of the polypeptide chain, 29 were isolated and substantially sequenced. The sequences obtained were valuable in providing overlaps for the alignment of about two-thirds of the sequences found in tryptic peptides [Wootton, J. C., Taylor, J, G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 739-748]. The blocked N-terminal peptide of the protein was isolated. This peptide was sequenced by mass spectrometry, and found to have N-terminal N-acetylserine by Howard R. Morris and Anne Dell, whose results are presented as an Appendix to the main paper. The staphylococcal proteinase showed very high specificity for glutamyl bonds in the NH4HCO3 buffer used. Partial splits of two aspartyl bonds, both Asp-Ile, were probably attributable to the proteinase. No cleavage of glutaminyl or S-carboxymethylcysteinyl bonds was found. Additional experimental detail has been deposited as Supplementary Publication SUP 50053 (5 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K, from whom copies may be obtained under the terms given in Biochem. J. (1975) 1458 5. 相似文献
16.
17.
The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press]. 相似文献
18.
The two cysteinyl residues present in histidine decarboxylase from Lactobacillus 30a differ greatly in reactivity. One (class 1) reacts readily in the native state with dithiobis-(2-nitrobenzoate) with complete loss of enzyme activity; the other (class 2) reacts only after denaturation of the enzyme (Lane, R. S., and Snell, E. E. (1976) Biochemistry 15, 4175-4179). These differences in reactivity permitted use of covalent (disulfide) chromatography to isolate separate peptides that contain these two residues. Sequence analysis showed that the class 1 cysteinyl residue is at position 147 in a hydrophilic portion of the alpha chain (Huynh, Q. K., Recsei, P. A., Vaaler, G. L., and Snell, E. E. (1984) J. Biol. Chem. 259, 2833-2839), while the class 2 cysteinyl residue is present at position 71, adjacent to a hydrophobic portion of the same chain. Cysteinyl peptides identical with or homologous to the class 2 cysteinyl peptide of the Lactobacillus 30a enzyme were isolated from the alpha subunits of histidine decarboxylases from Lactobacillus buchneri and Clostridium perfringens, respectively. The L. buchneri enzyme also contained a peptide homologous to the class 1 cysteinyl peptide from Lactobacillus 30a. However, no corresponding peptide was present in the enzyme from C. perfringens, in which the second cysteinyl residue of the alpha chain occupies position 3, very near the essential pyruvoyl residue. This enzyme, unlike those from Lactobacillus 30a or L. buchneri, also contains one cysteinyl residue in its beta chain. Although Cys 147 is an active site residue in histidine decarboxylase from Lactobacillus 30a, the absence of a corresponding residue in the C. perfringens enzyme confirms previous indications (Recsei, P. A., and Snell, E. E. (1982) J. Biol. Chem. 257, 7196-7202) that this SH group is not essential for decarboxylase action. 相似文献
19.
Acetylation of prothrombin fragment 1 in acetate-borate buffer at pH 8.5 resulted in the appearance of increased light absorbance at about 250 nm. Protease digestions resulted in isolation of a single peptide (residues 94-99) with intense absorbance at about 250 nm (estimated extinction coefficient of 5000 M-1 cm-1). Amino acid analysis showed the expected composition except for the absence of His-96. Instead, an unidentified amino acid which had a ninhydrin product with absorption properties similar to those of proline eluted near aspartate. When sequenced, this peptide (YP?KPE containing epsilon-amino-acetyllysine) lacked histidine at the third position but gave a high yield of a PTH derivative that eluted near PTH-Gly from the HPLC column. Fast atom bombardment mass spectrometry of the derivatized 94-99 peptide showed a mass that was 74 units higher than expected. The histidine degradation product was identified as a di-N-acetylated side chain with an opened imidazole ring and loss of C2 of the ring. While a similar degradation pattern has previously been reported during acylation of histidine, the high chemical reactivity exhibited by His-96 was unusual. For example, under conditions sufficient for quantitative derivatization of His-96, His-105 of fragment 1 was not derivatized to a detectable level. Furthermore, His-96 in fragment 1 was at least an order of magnitude more susceptible to degradation than His-96 in the isolated 94-99 peptide. His-96 is therefore one of several neighboring amino acids of the kringle portion of fragment 1 that displays highly unusual chemistry (see also Asn-101 [Welsch, D.J., & Nelsestuen, G. L. (1988) Biochemistry 27 4946-4952] and Lys-97 [Pollock, J.S., Zapata, G.A., Weber, D.J., Berkowitz, P., Deerfield, D.W., II, Olson, D.L., Koehler, K.A., Pedersen, L.G., & Hiskey, R.G. (1988) in Current Advances in Vitamin K Research (Suttie, J.W., Ed.) pp 325-334, Elsevier Science, New York]).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
M L Speranza G Valentini P Iadarola M Stoppini M Malcovati G Ferri 《Biological chemistry Hoppe-Seyler》1989,370(3):211-216
Three peptides containing 6-pyridoxyllysine have been isolated from the tryptic digest of the allosteric fructose-1,6-bisphosphate-dependent pyruvate kinase from Escherichia coli, which had been almost completely inactivated with pyridoxal 5'-phosphate. The labelled peptides have been sequenced. The comparison of their sequences with the primary structure of the cat muscle pyruvate kinase allowed to state that peptide I fits the region spanning residues 423-438 (53% identity), peptide II corresponds to residues 442-457 (44% identity) and peptide III encompasses residues 342-368 (70% identity). These findings are discussed in connection with our previous results on the involvement of the three peptides in the catalytic and regulatory properties of the enzyme (Valentini, G., Speranza, M.L., Iadarola, P., Ferri, G. & Malcovati, M. (1988) Biol. Chem. Hoppe-Seyler 369, 1219-1226) and in connection with their location in the three-dimensional structure of the cat muscle pyruvate kinase (Muirhead, H., Clayden, D.A., Lorimer, C.G., Fothergill-Gilmore, L.A., Schiltz, E. & Schmitt, W. (1986) EMBO J. 5, 475-481). 相似文献