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1.
C Fabris D Basso L Benini T Meggiato G Del Favero G Cavallini M P Panozzo P Fogar C Angonese I Vantini 《Enzyme》1989,42(2):80-86
Serum and urine elastase 1, its renal output and clearance and urinary gamma-glutamyltransferase and ribonuclease excretions were measured in 16 patients with pancreatic cancer, 23 with chronic pancreatitis and in 22 healthy controls in order to evaluate elastase 1 plasma-urine transfer in chronic pancreatic disease and to investigate any factors that might influence the clearance of this enzyme. In an additional group of 17 patients with different pancreatic diseases the serum molecular size distribution of elastase 1 after chromatography was ascertained. An increased urinary elastase 1 output was found in 4/16 patients with pancreatic cancer and in 6/23 with chronic pancreatitis. No correlation was found between circulating elastase 1 and its urinary output; a negative correlation was detected between the serum levels of this enzyme and its clearance. The excretion of ribonuclease and gamma-glutamyltransferase was correlated with elastase 1 output and clearance. While the majority of elastase 1 in serum was accounted for by high molecular forms, probably the expression of complexes with serum inhibitors, free circulating enzyme was present in all patients with high serum elastase 1. Our findings suggest that elastase 1 urinary excretion increases in some patients with chronic pancreatic disease regardless of the neoplastic or inflammatory nature of the illness. Although the availability of different amounts of ultrafiltrable enzyme may play a role in influencing elastase 1 plasma-urine transfer, renal tubular damage appears to be the most important factor influencing the increase in the urinary output of elastase 1. 相似文献
2.
Qamar AA Burke SK Lafleur JD Ding BC Bland KS Wong MD Gustafson PN Blair AT Franano FN 《Biotechnology and applied biochemistry》2012,59(1):22-28
PRT-201 is a recombinant human pancreatic elastase under development as a treatment for blood vessels to promote hemodialysis access patency. Proteases such as elastase are normally inactivated by antiproteases such as alpha 1-antitrypsin. It is unknown if serum from patients with alpha 1-antitrypsin deficiency will inhibit PRT-201 elastase activity. An assay for PRT-201 elastase activity in the presence of serum was developed and validated. PRT-201 elastase activity inhibition curves were developed using serum and also using purified alpha 1-antitrypsin and alpha 2-macroglobulin. Serum from 15 patients with documented alpha 1-antitrypsin deficiency, some of whom were receiving alpha 1-antitrypsin augmentation therapy, and four normal volunteers was analyzed. Serum from normal volunteers and patients with alpha 1-antitrypsin deficiency completely inactivated PRT-201 elastase activity in vitro. In the alpha 1-antitrypsin-deficient patients, the volume of serum necessary to inhibit elastase activity was related to the serum concentration of alpha 1-antitrypsin and augmentation therapy. Purified alpha 1-antitrypsin and alpha 2-macroglobulin were each alone capable of completely inhibiting PRT-201 elastase activity. It is unlikely that the use of PRT-201 will be associated with negative outcomes in patients with alpha 1-antitrypsin deficiency. 相似文献
3.
Immunoglobulin G subclasses of anti-thyroid peroxidase autoantibodies in human autoimmune thyroid diseases 总被引:1,自引:0,他引:1
A distribution of immunoglobulin G (IgG) subclass of anti-thyroid peroxidase (TPO) autoantibodies was studied to know whether anti-TPO autoantibodies are closely implicated in the pathogenesis of human autoimmune thyroid diseases. As a result of analyzing 14 patients' sera, 7 with Graves' disease and 7 with Hashimoto's thyroiditis, anti-TPO autoantibodies were found to consist of mainly IgG1 subclass. Percentages of both IgG1 and IgG2 subclasses in IgG class of autoantibodies corresponded to those in the normal serum composition, whereas IgG3 subclass was scarcely contained in anti-TPO autoantibodies and IgG4 subclass markedly increased. It was thought that anti-TPO autoantibodies had a capability to lyse thyroid follicular cells by the mechanism of antibody-dependent complement-mediated cytolysis, because IgG1 and IgG2 subclasses of antibodies can fix complement and TPO locates in apical membrane surface of thyroid follicular cells. Comparing Graves' disease with Hashimoto's thyroiditis, mean percentages of both IgG1 and IgG2 subclasses of 2 groups were statistically different. Namely, sera of patients with Graves' disease had higher and lower mean percentages of IgG1 and IgG2 subclasses of autoantibodies, respectively, than those with Hashimoto's thyroiditis, though no plausible explanation for these differences can be offered at the present time. 相似文献
4.
Hypocomplementemic urticarial vasculitis syndrome (HUVS) is an apparent autoimmune disorder that resembles SLE. We previously showed that C1q precipitins in HUVS sera are IgG autoantibody to human C1q. We have compared HUVS anti-C1q autoantibody to a similar autoantibody in the serum of some patients with SLE. As with anti-C1q autoantibody in SLE sera, the HUVS autoantibody binds only to the collagen-like region (CLR) of C1q. In both HUVS and SLE, IgG2 is the predominant subclass of IgG autoantibody and IgM autoantibody to C1q is uncommon. In both diseases, anti-C1q autoantibodies bind preferentially to surface-adsorbed C1q or CLR fragments compared to these antigens in solution. Finally, when HUVS or SLE autoantibodies were added to CLR-coated wells already bound, respectively, by SLE or HUVS autoantibodies, no increases in CLR binding were observed, suggesting that HUVS and SLE autoantibodies to C1q bind to the same CLR epitope(s). 相似文献
5.
An elastase from pig aorta has been partially purified and characterised; it exhibits immunological cross reaction with pig pancreatic elastase. Its proteolytic (k-elastin gel and polymeric elastin substrates) and esterolytic (N-succinoyl-trialanine paranitroanilide) activities as well as its degree of inhibition by serum protease inhibitors (alpha1-antitrypsin and alpha2-macro-globulin) differ sensibly from those of pancreatic elastase [14,16]. 相似文献
6.
Trace amounts of porcine pancreatic elastase mixed with porcine serum, or injected intravenously into the pig, were found to be bound mainly to alpha 1- and alpha 2-macroglobulin (90%). Alpha 1-macroglobulin approached saturation with elastase before significant binding to alpha 2-macroglobulin was demonstrable. Human pancreatic cationic elastase showed in human serum preferential binding to alpha 2-macroglobulin, but the elastase was also bound by alpha 1-protease inhibitor and by alpha 1-antichymotrypsin. The porcine elastase-alpha 1 alpha 2-macroglobulin complexes injected intravenously or formed in vivo in the pig were rapidly eliminated from the blood stream following a first order reaction with t 1/2 = 8 min. Porcine alpha 1-protease-inhibitor-bound elastase disappeared considerably more slowly. 相似文献
7.
Y Shirasu H Yoshida T Mikayama S Matsuki J Tanaka H Ikenaga 《Journal of biochemistry》1986,99(6):1707-1712
We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum. 相似文献
8.
Substrate specificity of human pancreatic elastase 2 总被引:4,自引:0,他引:4
The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied. 相似文献
9.
This paper is a continuation of our study of various animal pancreatic enzymes which are related to human pancreatic elastase 1 (Sziegoleit, A. & Linder, D. (1986) Biol. Chem. Hoppe-Seyler, 367, 527-531). The isolation and immunological analysis of the related protein from bovine pancreas disclosed that the third subunit of the procarboxypeptidase A complex is the antibody-binding component. The similarity of this subunit to elastase 1 is affirmed by comparison of their primary structures. While the complete amino-acid sequence of bovine subunit III recently has been published (Venot, N., Sciaky, M., Puigserver, A., Desnuelle, P. & Laurent, G. (1986) Eur. J. Biochem. 157, 91-99), we here present the amino-acid sequence of the carboxy-terminal tryptic peptide of human pancreatic elastase 1 showing a high degree of homology. 相似文献
10.
E L Gustavsson K Ohlsson A S Olsson 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1980,361(2):169-176
1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive. 相似文献
11.
Deloumeau A Bayard S Coquerel Q Déchelotte P Bole-Feysot C Carlander B Cochen De Cock V Fetissov SO Dauvilliers Y 《PloS one》2010,5(10):e13320
Background
Hypocretin peptides participate in the regulation of sleep-wake cycle while deficiency in hypocretin signaling and loss of hypocretin neurons are causative for narcolepsy-cataplexy. However, the mechanism responsible for alteration of the hypocretin system in narcolepsy-cataplexy and its relevance to other central hypersomnias remain unknown. Here we studied whether central hypersomnias can be associated with autoantibodies reacting with hypocretin-1 peptide present as immune complexes.Methodology
Serum levels of free and dissociated (total) autoantibodies reacting with hypocretin-1 peptide were measured by enzyme-linked immunosorbent assay and analyzed with regard to clinical parameters in 82 subjects with narcolepsy-cataplexy, narcolepsy without cataplexy or idiopathic hypersomnia and were compared to 25 healthy controls.Principal Findings
Serum levels of total but not free IgG autoantibodies against hypocretin-1 were increased in narcolepsy-cataplexy. Increased levels of complexed IgG autoantibodies against hypocretin-1 were found in all patients groups with a further increase in narcolepsy-cataplexy. Levels of total IgM hypocretin-1 autoantibodies were also elevated in all groups of patients. Increased levels of anti-idiotypic IgM autoantibodies reacting with hypocretin-1 IgG autoantibodies affinity purified from sera of subjects with narcolepsy-cataplexy were found in all three groups of patients. Disease duration correlated negatively with serum levels of hypocretin-1 IgG and IgM autoantibodies and with anti-idiotypic IgM autoantibodies.Conclusion
Central hypersomnias and particularly narcolepsy-cataplexy are characterized by higher serum levels of autoantibodies directed against hypocretin-1 which are present as immune complexes most likely with anti-idiotypic autoantibodies suggesting their relevance to the mechanism of sleep-wake cycle regulation. 相似文献12.
C R L Graves J F R Robertson A Murray M R Price C J Chapman 《The journal of peptide research》2005,66(6):357-363
Numerous reports document the existence of autoantibodies to MUC1 in the circulation of individuals with breast and other solid malignancies, with the majority of researchers utilizing MUC1 peptides in their detection. This report documents the purification, using peptide and whole molecule, and characterization of such autoantibodies from an individual with an unusual, highly MUC1-positive, myosarcoma. Purification of autoantibodies from serum was performed using affinity chromatography against either MUC1 peptide or whole molecule MUC1 [derived both from the patient (Pt-MUC1) and from a pool of sera from patients with advanced breast cancer (ABC-MUC1)]. Enzyme-linked immunosorbent assays (ELISAs) were used to compare specificity of purified autoantibodies. Peptide epitopes were determined by Ptifcan system against 7-mer peptides covering the 20 amino acid repeat of the MUC1 extracellular domain. Substantially higher amounts of autoantibodies were isolated when purifying against Pt-MUC1 rather than either ABC-MUC1 or peptide. Whole molecule purified autoantibodies demonstrated an increased specificity for tumour-derived MUC1. Pt-MUC1 autoantibodies were of both the immunoglobulin (Ig)G and IgM class, whilst autoantibodies purified against ABC-MUC1 and MUC1 peptide were IgG only. A greater range of peptide epitopes was defined by those autoantibodies purified against whole molecule. This report presents data indicating the presence of autoantibodies to MUC1 in an individual diagnosed with a MUC1 over-expressing myosarcoma. Confirmation of these autoantibodies as being specific for tumour-associated MUC1 is given. Further, it suggests that, although autoantibodies are present that recognize core protein determinants, the initial, and dominant, immunizing epitope is not purely pretentious in nature. 相似文献
13.
The IgG3 subclass of β1-adrenergic receptor autoantibodies is an endogenous biaser of β1AR signaling
Maradumane L. Mohan Yuji Nagatomo Prasenjit Prasad Saha Sromona D. Mukherjee Timothy Engelman Rommel Morales Stanley L. Hazen W. H. Wilson Tang Sathyamangla V. Naga Prasad 《Molecular biology of the cell》2021,32(7):622
Dysregulation of immune responses has been linked to the generation of immunoglobulin G (IgG) autoantibodies that target human β1ARs and contribute to deleterious cardiac outcomes. Given the benefits of β-blockers observed in patients harboring the IgG3 subclass of autoantibodies, we investigated the role of these autoantibodies in human β1AR function. Serum and purified IgG3(+) autoantibodies from patients with onset of cardiomyopathy were tested using human embryonic kidney (HEK) 293 cells expressing human β1ARs. Unexpectedly, pretreatment of cells with IgG3(+) serum or purified IgG3(+) autoantibodies impaired dobutamine-mediated adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) generation while enhancing biased β-arrestin recruitment and Extracellular Regulated Kinase (ERK) activation. In contrast, the β-blocker metoprolol increased AC activity and cAMP in the presence of IgG3(+) serum or IgG3(+) autoantibodies. Because IgG3(+) autoantibodies are specific to human β1ARs, non–failing human hearts were used as an endogenous system to determine their ability to bias β1AR signaling. Consistently, metoprolol increased AC activity, reflecting the ability of the IgG3(+) autoantibodies to bias β-blocker toward G-protein coupling. Importantly, IgG3(+) autoantibodies are specific toward β1AR as they did not alter β2AR signaling. Thus, IgG3(+) autoantibody biases β-blocker toward G-protein coupling while impairing agonist-mediated G-protein activation but promoting G-protein–independent ERK activation. This phenomenon may underlie the beneficial outcomes observed in patients harboring IgG3(+) β1AR autoantibodies. 相似文献
14.
A Sziegoleit D Linder M Schlüter M Ogawa S Nishibe K Fujimoto 《European journal of biochemistry》1985,151(3):595-599
The proteolytic attack of the cholesterol-binding pancreatic proteinase (CBPP) on the oxidized insulin A and B chains as well as on glucagon was investigated by kinetic studies. The reaction products were isolated by high-pressure liquid chromatography and identified by amino acid analysis. The combined results reveal a pronounced selectivity of CBPP for the peptide bonds at the carboxy ends of Ala, Val, Leu, Ser, His and Thr residues with Ala, Val and Leu most favoured, indicating a close catalytic relationship to porcine pancreatic elastase [Narayanan, A. S. & Anwar, R. A. (1969) Biochem. J. 114, 11-17] and the anionic porcine pancreatic protease E [Kobayashi R., Kobayashi, Y. & Hirs, C. H. W. (1981) J. Biol. Chem. 256, 2460-2465] which resembles human pancreatic elastase 1. The immunological comparison indeed disclosed the identity of CBPP with human pancreatic elastase 1. 相似文献
15.
Irina B. Magorivska Rostyslav O. Bilyy Anna M. Havrylyuk Valentyna V. Chop'yak Rostyslav S. Stoika Yury Y. Kit 《Journal of molecular recognition : JMR》2010,23(5):495-502
Novel hydrolytic activity of the anti‐histone H1 antibodies (Ab) toward histone H1 and myelin basic protein (MBP) was shown. Blood serum of ten patients with clinically diagnosed systemic lupus erythematosus (SLE), and nine healthy donors (control) were screened for the anti‐histone H1 antibody‐ and anti‐MBP antibody‐mediated specific proteolytic activity. IgGs were isolated by chromatography on Protein G‐Sepharose, and four of ten SLE patients appeared to possess IgGs that were capable of cleaving both histone H1 and MBP. Such activity was confirmed to be an intrinsic property of the IgG molecule, since it was preserved at gel filtration at alkaline and acidic pH. At the same time, proteolytic activity was absent in the sera‐derived Ab of all healthy donors under control. Anti‐histone IgGs were purified by the affinity chromatography on histone H1‐Sepharose. Their cross‐reactivity toward cationic proteins (histones, lysozyme, and MBP) and their capability of hydrolyzing histone H1 and MBP were detected. However, these IgGs were not cleaving core histones, lysozyme, or albumin. Capability of cleaving histone H1 and MBP was preserved after additional purification of anti‐histone H1 IgGs by the HPLC gel filtration. The protease activity of anti‐histone H1 IgG Ab was inhibited by serine protease inhibitors. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
16.
O A Vartanian 《Molekuliarnaia biologiia》1991,25(4):1033-1039
Sera of patients bearing autoimmune diseases (rheumatoid arthritis and systemic lupus erythematosus) and sera of clinically healthy donors were examined by ELISA for the presence of autoantibodies against tryptophanyl-, tyrosyl- and phenylalanyl-tRNA synthetases. Pure bovine synthetases served as antigens. It was shown that in patients with both autoimmune diseases all three enzyme autoantibodies were revealed at serum dilution 1/1600-1/3200. Moreover, by means of monoclonal antibodies against the same enzymes used for immunoaffinity sorption, antiidiotypic antibodies of IgG type against autoantibodies were detected. A conclusion has been made that autoimmune diseases are characterized by autoimmune response for many aminoacyl-tRNA synthetases irrespectively of their quaternary structure, intracellular location etc both at the level of primary and secondary antibodies. 相似文献
17.
C Fabris G Del Favero A Panucci M Plebani F Di Mario A Piccoli D Basso A Burlina R Naccarato 《Enzyme》1986,35(2):82-86
In order to investigate modifications of serum levels of elastase 1, immunoreactive trypsin, alpha 1-antitrypsin and alpha 2-macroglobulin in chronic pancreatic disease, and to speculate on the possible relationships among these parameters, the enzymes and inhibitors were assayed in the sera of 33 control subjects, 34 pancreatic cancer, 28 chronic pancreatitis and 36 extra-pancreatic diseases. An increase of elastase 1, alpha 1-antitrypsin and alpha 2-macroglobulin was detected in pancreatic cancer, chronic pancreatitis and extra-pancreatic diseases; no changes were found for serum immunoreactive trypsin. Multiple regression analyses showed that only 7% of elastase 1 was explained by inhibitors with alpha 1-antitrypsin playing a major role. Inhibitors did not influence immunoreactive trypsin. Our data indicate that the variations of the serum levels of proteases and antiproteases in chronic pancreatic disease are probably independent of each other. 相似文献
18.
Comparison between autoantibodies arising during Trypanosoma cruzi infection in mice and natural autoantibodies 总被引:6,自引:0,他引:6
T Ternynck C Bleux J Gregoire S Avrameas C Kanellopoulos-Langevin 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(4):1504-1511
The autoantibodies induced in (C57BL/6 x BALB/c)F1 mice during Trypanosoma cruzi (CL strain) infection were analyzed and compared with natural autoantibodies present in healthy mice. Mice were killed at intervals after infection and their sera were tested by enzyme immunoassay against a panel of self- and non-self-Ag: actin, myoglobin, myosin, tubulin, DNA, and TNP-OVA. The level of IgM and IgG autoantibodies against all Ag started to increase from day 15 until 6 wk after the parasite infection. The high level of all autoantibodies persisted 3 mo postinfection, and 1 yr later, half of the mice still had elevated levels of IgM and IgG autoantibodies, particularly antitubulin IgG antibodies. IgM and IgG were isolated from pools of normal and infected mouse sera and their binding capacity to all Ag was compared. The titers of infected mouse sera were increased and the slopes of both IgM and IgG binding curves of autoantibodies to actin, myosin, and tubulin were greater than those of control mouse sera, indicating higher affinities. The average dissociation constant of the IgG2a autoantibody to mouse tubulin was 5 times lower than that of natural antitubulin IgG2a antibodies. Furthermore, absorption of the IgG from infected mouse sera onto a tubulin immunoadsorbent removed half the reactivity with tubulin and also with myosin, actin and parasite extracts. The eluted antibodies bound the same Ag. When IgG were further analyzed by Western blot on proteolytic fragments of tubulin, we found that antibodies from both groups bound to the same broad spectrum of polypeptide bands. However, additional fragments were recognized by antibodies from infected mice. All these results indicate that the autoantibodies naturally present in mice are significantly affected after infection with T. cruzi, in quantity as well as in specificity and affinity. 相似文献
19.
Atta AM Sousa-Atta ML D'Oliveira A Almeida RP Araújo MI Carvalho EM 《Memórias do Instituto Oswaldo Cruz》2002,97(1):101-103
Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera. 相似文献
20.
Investigation of the active center of rat pancreatic elastase 总被引:1,自引:0,他引:1
J G Bieth S Dirrig M L Jung C Boudier E Papamichael C Sakarellos J L Dimicoli 《Biochimica et biophysica acta》1989,994(1):64-74
We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme. 相似文献