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1.
Tryptase from rat skin: purification and properties 总被引:2,自引:0,他引:2
Tryptase was purified 13,000-fold to apparent homogeneity from rat skin. The two-step procedure involved ammonium sulfate fractionation of the initial extract followed by combined sequential affinity chromatography on agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose. The purified enzyme had a specific activity toward N-benzoylarginine ethyl ester (BzArgOEt) of 170 mumol/min mg-1 and was obtained in a yield of 28% as determined by the specific substrate, H-D-Ile-Pro-Arg-p-nitroanilide. Rat skin tryptase was thermal labile, losing 50% of its activity when preincubated for 30 min at 30 degrees C. The presence of NaCl (1 M) improved thermal stability and was necessary for long-term storage. Heparin did not stabilize the enzyme against thermal denaturation, and heparin-agarose failed to bind the enzyme. Rat skin tryptase was inhibited by diisopropylphosphofluoridate, antipain, leupeptin, and aprotinin but not by alpha 1-antitrypsin, ovomucoid, or soybean or lima bean trypsin inhibitors. Substrate specificity studies using a series of tri- and tetrapeptidyl-p-nitroanilide and peptidyl-7-amino-4-methylcoumarin substrates demonstrated the existence of an extended substrate binding site. Rat skin tryptase hydrolyzed [Arg8]vasopressin, neurotensin, and the oxidized B-chain of insulin at the -Arg8-Gly9-NH2, -Arg8-Arg9-, and -Arg22-Gly23-bonds, respectively. No general proteinase activity was observed toward casein, hemoglobin, or azocoll. Rat skin tryptase had a Mr of 145,000 by gel filtration. The subunit Mr was either 34,000 or 30,000 depending on the electrophoretic technique used. Treatment of the enzyme with peptide N-glycosidase F (N-glycanase) decreased the subunit Mr by 4000. The enzyme exhibited multiple isoelectric forms (pI's of 4.5-4.9). Rat skin tryptase was found to be related statistically to other tryptases on the basis of amino acid composition. The N-terminal amino acid sequence was Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Trp12-Pro13- Trp14- Gln15-Val16-Ser17-Leu18-Arg19-Val20- --21-Asp-22Thr23-Tyr24-Typ25-, with a putative glycosylation site at residue 21. This sequence was 72-80% homologous with the N-terminus of other tryptases but only 40% homologous with that of bovine trypsin. 相似文献
2.
A post-proline cleaving enzyme [prolyl endopeptidase, EC 3.4.21.26] was purified about 3,700-fold from an extract of bovine brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and PCMB-T-Sepharose, and gel filtration on Sephadex G-200 using N-carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap), thyrotropin releasing hormone (TRH) and oxytocin as substrates. The purified enzyme appeared homogeneous as judged by disc gel and SDS gel electrophoreses. The enzyme was most active at pH 7.5 and 7.2 with Z-Gly-Pro-2-NNap and TRH, respectively, and hydrolyzed peptide bonds involving Pro-X (X=amino acid, peptide, ester and amide) bonds of synthetic substrates, oxytocin, vasopressin, neurotensin, substance P, tuftsin, bradykinin, and insulin B chain. However, the enzyme was inert toward collagen, gelatin, and casein. The enzyme was completely inactivated by diisopropylphosphorofluoridate (DFP), Z-Gly-Pro-chloromethyl ketone and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenylmethane sulfonylfluoride (PMSF) or metal chelators. Determination of the amino acid composition revealed that the enzyme contained 25 half-cystines. Modification of three cysteine residues of the enzyme by PCMB led to complete inactivation. The isoelectric point of the enzyme was 4.8, and the molecular weight was estimated to be 76,000 by ultracentrifugal analysis and 75,000-74,000 by both gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is present as a monomer. These results indicate that the post-proline cleaving enzyme from bovine brain is very similar to those previously purified from lamb brain and kidney in its enzymatic properties, substrate specificity and physicochemical properties, in sharp contrast with the results obtained by Tate, who reported that the bovine brain prolyl endopeptidase was inert toward oxytocin, vasopressin and bradykinin. 相似文献
3.
Experiments were performed on isolated salt-perfused rat lungs to determine the receptor type(s) responsible for the pulmonary vascular effects of the neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin. Bolus administration of AVP to lungs preconstricted with the thromboxane mimetic U-46619 resulted in a dose-dependent vasodilatory response (approximately 65% reversal of U-46619-induced vasoconstriction at the highest dose tested) that was blocked by pretreatment with a selective V1- but not by a selective V2-vasopressinergic receptor antagonist. Administration of a selective V1-agonist to the preconstricted pulmonary vasculature resulted in a vasodilatory response similar to that observed with AVP (approximately 55% reversal of U-46619 vasoconstriction), which was blocked by prior administration of the selective V1-receptor antagonist. Administration of the selective V2-receptor agonist desmopressin to the preconstricted pulmonary vasculature resulted in a small (approximately 8% reversal of U-46619 vasoconstriction) vasodilatory response that was, nevertheless, greater than that produced by addition of vehicle alone and was attenuated by pretreatment with a selective V2-receptor antagonist. Finally, oxytocin also caused vasodilation in the preconstricted pulmonary vasculature; however, the potency of oxytocin was approximately 1% of AVP, and the vasodilation produced by oxytocin was blocked by prior administration of a selective V1-receptor antagonist, suggesting that oxytocin acts via V1-vasopressinergic receptor stimulation. We conclude from these experiments that AVP and oxytocin dilate the preconstricted pulmonary vasculature primarily via stimulation of V1-vasopressinergic receptors. V2-receptor stimulation results in a minor vasodilatory response, although its physiological significance is unclear. 相似文献
4.
The lysyl ester analogue p-nitrophenyl alpha-amino-p-toluate hydrobromide was synthesized, and its reactions with thrombin, trypsin, and plasmin were studied by stopped-flow and conventional methods. Kinetic parameters were compared with those determined for the arginyl ester analogue, p-nitrophenyl p-guanidinobenzoate hydrochloride, with these enzymes. By following nitrophenol release or proflavin absorption changes in the stopped-flow spectrophotometer, the constants Ks (enzyme-substrate binding), k2 (acylation), and k3 (deacylation) were determined. The major findings were: (1) Ks values were similar regardless of the substrate or the enzyme; (2) k3 was approximately the same for the reaction of the lysyl ester analogue with any enzyme; (3) k2 for the lysyl ester analogue was 1100 times greater with trypsin than with thrombin; and (4) k2 with thrombin was 60 times greater for the arginyl than for the lysyl ester analogue. The results suggest that the limited cleavage of lysyl bonds by thrombin is due in part to restricted acylation rather than substrate binding. The active site of thrombin, compared with that of trypsin, appears to have a more stringent requirement for the spatial relationship between the cationic group and the bond cleaved in substrates. 相似文献
5.
W D Schleuning R Hell H Schiessler H Fritz 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1975,356(12):1915-1921
Boar acrosin was isolated to electrophoretic homogeneity by modification of the method described by us previously. Titration with p-nitrophenyl p-guanidinobenzoate revealed a molar activity of 8900 toward BzArgOEt and of 780 towards BzArgNHNp. Treatment with neuraminidase led to an altered electrophoretic mobility, indicating that acrosin is sialogycoprotein 相似文献
6.
A proteinase was purified from resting seeds of Cucurbita ficifolia by ammonium sulfate fractionation and successive chromatography on CM-cellulose, Sephacryl S-300 and TSK DEAE-2SW (HPLC) columns. Inhibition by DFP and PMSF suggests that the enzyme is a serine proteinase. The apparent molecular mass of this enzyme is ca. 77 kDa. The optimum activity for hydrolysis of casein and Suc-Ala-Ala-Pro-Phe-pNA is around pH 10.5. The following peptide bonds in the oxidized insulin B-chain were hydrolysed by the proteinase: Phe1-Val2, Asn3-Gln4, Gln4-His5, Cya7-Gly8, Glu13-Ala14, Ala14-Leu15, Cya19-Gly20, Pro28-Lys29 and Lys29-Ala30. The proteinase is more selective towards the native squash seed trypsin inhibitor (CMTI I) and primarily cuts off only its N-terminal arginine. The inhibitor devoided of the N-terminal arginine residue is still active against trypsin. 相似文献
7.
A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column. The sperminogen eluted from the column in a single band that was completely separated from the proacrosin band. This separation was confirmed by a gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymograph. This zymograph also demonstrated that the final sperminogen preparation contained four forms of zymogen, with molecular weights between 32,000 and 36,000. At neutral pH, the sperminogen was converted into spermin, its enzymatically active form, yielding a sigmoidal curve typical of zymogen autoactivation. The effects of several factors on the rate of this autoconversion indicate specific differences between sperminogen and proacrosin. Spermin hydrolyzed N-alpha-benzoyl-L-arginine ethyl ester (BzArgOEt), and was inhibited by lima bean trypsin inhibitor, pancreatic trypsin inhibitor, N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), and tosyl-L-lysine chloromethyl ketone, indicating that the enzyme has a trypsin-like specificity and probably belongs to the class of trypsin-like enzymes. Since acrosin is generally believed to be the only trypsin-like enzyme in mammalian sperm, the demonstration of human sperminogen and spermin necessitates further inquiry into the functions and the relationships between sperm proteinase systems. 相似文献
8.
P Cottin J L Azanza P Vidalenc A Ducastaing C Valin A Ouali 《Reproduction, nutrition, development》1981,21(2):309-317
This paper describes the isolation, purification and properties of a specific inhibitor of calcium-activated neutral proteinase (CaANP) in rabbit skeletal muscle. The inhibitor was a thermo-acid-stable protein degraded by trypsin and chymotrypsin and seemed to contain two polypeptide chains with molecular weights of 70 000 and 13 000 daltons. Maximal inhibitory activity was obtained at neutral pH. High salt concentrations were needed to suppressinhibition. Inhibitor concentration had no effect on the optimal Ca++ ion levels for CaANP. These experiments also show that enzyme inhibitor association was instantaneous and did not need any incubation. 相似文献
9.
Partial purification and characterization of a neutral protease which cleaves type IV collagen 总被引:11,自引:0,他引:11
A neutral protease has been extracted from the media of cultured metastatic tumor cells and purified approximately 1000 times after sequential ammonium sulfate fractionization, concanavalin A column chromatography, and molecular sieve chromatography. The protease has an apparent molecular weight of 70 000--80 000, is inactive at acid pH, requires trypsin activation, and is inhibited by ethylene-diaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide, or soybean trypsin inhibitor. The enzyme produces specific cleavage products for both chains of pro type IV collagen isolated without pepsinization and apparently cleaves at one point in a major pepsin-extracted chain of placenta type IV collagen. The partially purified enzyme fails to significantly degrade other collagens or fibronectin under digestion conditions in which specific reaction products are produced for type IV collagen. The existence of this enzyme is significant since previously described animal collagenases fail to degrade type IV collagen. Such a type IV specific collagenase could play a role in tumor invasion and may be secreted by other cells such as endothelial cells, epithelial cells, and immune cells. 相似文献
10.
A proteolytic enzyme, which causes the limited degradation of cardiac myosin, was purified from rat heart myofibrils. The purified enzyme (a myosin-cleaving protease) was apparently homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Autolysis of the purified enzyme was observed at neutral pH without high concentration of CaCl2. The molecular weight was estimated to be 26 000-27 000. The enzyme was active against casein, N-acetyl-L-tyrosine ethyl ester and N-glutaryl-L-phenylalanine-4-nitroanilide (Glu-Phe-NAn), but less active with N-benzoyl-DL-arginine-4-nitroanilide. Optimum pH values for the enzyme were 9.0 for casein and 8.4 for Glu-Phe-NAn. Caseinolytic activity of the enzyme was completely inhibited with phenylmethylsulfonyl fluoride and diisopropylphosphofluoride and partially inhibited with L-1-tosyl-L-phenylalanine chloromethyl ketone (Tos-PheCH2Cl) and soybean trypsin inhibitor. Tos-LysCH2Cl had no effect. Sulfhydryl reagents, metal-chelating agents and metal ions except for Zn2+ had little or no effect on the activity. Degradation of cardiac myosin with the enzyme produced two fragments having molecular weights of 130 000 and 94 000, accompanied by the disappearance of myosin heavy chain and light chain 2. Myosin degradation with the enzyme was more restrictive than with chymotrypsin. 相似文献
11.
M Nishikata 《Journal of biochemistry》1984,95(4):1169-1177
An enzyme was purified from soybean seeds mainly by repeated ion-exchange chromatography using benzoyl-L-arginine p-nitroanilide (BAPA) as a substrate. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The molecular weight was estimated as 59,000 by gel filtration. The enzyme was most active toward BAPA between pH 8 and 10. The enzyme was inactive toward protein substrates but hydrolyzed synthetic substrates and oligopeptides exclusively at the carboxyl side of L-arginine and L-lysine. Kinetic studies using synthetic substrates showed that, on the basis of Vmax/Km, the enzyme preferentially hydrolyzed amide substrates over ester substrates. Benzoyl-L-arginine 4-methylcoumaryl-7-amide (Bz-Arg-MCA) was the best substrate. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl), leupeptin, and antipain. p-Chloromercuribenzoate (PCMB) was only partially inhibitory. Various protein inhibitors of trypsin such as soybean trypsin inhibitor were ineffective. From the primary specificity and susceptibility to chemicals, the enzyme can be said to be a trypsin-like serine protease. Although the physiological role of the enzyme is unclear, it seems likely that it is involved in limited hydrolysis of certain physiological peptides during processing. 相似文献
12.
Cheung AW Danho W Swistok J Qi L Kurylko G Rowan K Yeon M Franco L Chu XJ Chen L Yagaloff K 《Bioorganic & medicinal chemistry letters》2003,13(1):133-137
Systematic substitution of His(6) residue using non-selective hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)) as the template led to the identification of Bu-Atc(6)(2-aminotetraline-2-carboxylic acid)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which showed moderate selectivity towards hMC4R over hMC1R. Further SAR studies resulted in the discovery of Penta-5-BrAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) and Penta-5-Me(2)NAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which are potent hMC4R agonists and are inactive in hMC1R, hMC3R and hMC5R agonist assays. 相似文献
13.
Twenty days after bilateral adrenalectomy (ADX) or immediately after the last of three 6-h long immobilization periods, the levels of hypothalamic and neurohypophyseal L-[35S]Cys-labeled arginine vasopressin (AVP), oxytocin (OT), and somatostatin-14 (SRIF) (only stressed animals) were measured simultaneously in male Wistar rats, after third ventricular administration of the labeled precursor, via guide-cannulae. The acetic acid-extracted labeled peptide fractions were purified by two sequential HPLC steps. After a 4 h period of labeling, only L-[35S]Cys-AVP was selectively increased in the hypothalami of ADX-ized rats, compared to the sham-operated animals, possibly reflecting a significant activation of the paraventricular parvocellular (PVC) AVP/corticotropin-releasing factor (CRF) neurons. The increased accumulation of neurohypophyseal L-[35S]Cys-labeled AVP and OT in these animals, without changes in the endogenous levels of these peptides, as measured by UV absorbance, also suggests a moderate activation of the magnocellular (MGC) AVP and OT neurons, as a consequence of adrenal insufficiency. In response to immobilization stress, levels of L-[35S]Cys-OT were selectively increased in the hypothalami and corresponding neurohypophyses, 2 h and 4 h after receiving the label, concomitantly with a statistically significant reduction in the stores of OT in the neural lobes. AVP and SRJF biosynthesis remained unaffected by immobilization; the neurohypophyseal AVP stores likewise remained unchanged. These observations suggest the selective activation of MGC-OT neurons in response to chronic immobilization stress. Selective increases in hypothalamic L-[35S]Cys-AVP in ADX-ized rats, and in hypothalamic L-[35S]Cys-OT in chronically stress-immobilized rats, are presented as a measure of PVC-AVP/CRF and MGC-OT neuronal activation, respectively. 相似文献
14.
Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures. 总被引:32,自引:0,他引:32
Human skin procollagenase has been isolated, in pure form, from the medium of fibroblasts cultured in the presence or absence of added serum. Purification was achieved using a combination of cation-exchange (phosphocellulose or carboxymethylcellulose) and gel-filtration chromatography. Two forms (60 000 and 55 000 daltons) of the procollagenase were detected by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels and could be separated by chromatography on Ultrogel AcA-44. Each form was converted to active enzyme by trypsin, producing species of 50 000 and 45 000 daltons, respectively. An autoactivation process also occurred, which yielded active enzyme without a detectable change in molecular weight. Procollagenase also was found in organ cultures of human skin but only when serum was added to the medium. This suggests that a serum-inhibitable proteolytic system is present in these cultures which, like trypsin, converts procollagenase to the active enzyme forms that can be isolated from serum-free organ culture medium. The collagenase species obtained from either fibroblast or organ culture medium were chromatographically and electrophoretically identical. 相似文献
15.
Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs 总被引:1,自引:0,他引:1
A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm. 相似文献
16.
Neumann ID Torner L Toschi N Veenema AH 《American journal of physiology. Regulatory, integrative and comparative physiology》2006,291(1):R29-R36
In response to forced swimming (FS), AVP is released somato-dendritically within the supraoptic nucleus (SON) and paraventricular nucleus (PVN), but not from neurohypophyseal terminals into blood. Together with AVP, oxytocin (OXT) is released within the SON and PVN. Here, we studied the role of intra-SON and intra-PVN OXT in the regulation of local AVP release and into the blood in male rats. Within the SON, bilateral retrodialysis of an OXT receptor antagonist (OXT-A) increased local AVP release in response to FS [60 s, 21 degrees C, vehicle twofold, not significant (ns); OXT-A: 15-fold increase, P < 0.05] without significantly affecting basal AVP release. In addition, local OXT-A elevated plasma AVP secretion under basal conditions (twofold increase, P < 0.05) without further elevation after FS. Within the PVN, exposure to FS elevated local AVP release, reaching significance only in the OXT-A group (vehicle: 1.4-fold, ns; OXT-A: 1.6-fold increase, P = 0.050). Bilateral OXT-A into the PVN did not affect peripheral AVP secretion either under basal or stress conditions. Basal ACTH concentrations tended to be elevated by local OXT-A within the PVN (1.7-fold increase, P = 0.076). In contrast, the swim-induced ACTH secretion was attenuated after retrodialysis of OXT-A within both the SON (at 5 min) and PVN (at 15 min) (P < 0.05 both) compared with vehicle. The results demonstrate a receptor-mediated effect of OXT within the SON and PVN on local and neurohypophyseal AVP release, which depends upon the activity conditions. Further, while exerting an inhibitory effect on hypothalamo-pituitary-adrenal axis activity under basal conditions, hypothalamic OXT is essential for an adequate acute ACTH response. 相似文献
17.
A T-kininogenase has been purified to homogeneity from rat submandibular gland extracts by DEAE-Sepharose chromatography and preparative gel electrophoresis. The purified protein has an apparent Mr of 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and splits into heavy and light chains with Mr of 22,000 and 6,000 in the presence of dithiothreitol. It is an acidic glycoprotein with pI of 4.65-4.75. The carbohydrate moiety is located on the light chain and binds concanavalin A and wheat germ agglutinin. The active site serine residue of the heavy chain is labeled with [14C]diisopropylfluorophosphate and visualized by fluorography. NH2-terminal amino acid sequences of the light and heavy chains reveal 74-84% identity to rat tissue kallikrein, tonin, and other kallikrein-related enzymes. The enzyme cleaves T-kininogen to release T-kinin which was separated by high performance liquid chromatography on a reverse phase C18 column and identified by a kinin radioimmunoassay. Its T-kininogenase but not N-tosyl-L-arginine methyl ester esterase activity can be enhanced 10-fold in the presence of dithiothreitol. The esterolytic activity of the enzyme is inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, and antipain; whereas lima bean and ovomucoid trypsin inhibitors stimulate its activity. The enzyme is localized at the granular convoluted tubule and striated duct cells in rat submandibular glands by immunohistochemistry. The results indicate that T-kininogenase belongs to the group of structurally similar yet distinct kallikrein-like serine proteases. 相似文献
18.
Dimitrijevic L 《FEBS letters》1972,25(1):170-174
When Yeast lysyl-tRNA-ligase is extracted in the presence of protease inhibitor (PMSF) a homogeneous species of enzyme is obtained, the subunits of which have a mass of 70 000 daltons. Endogenous degradation produces lighter active species of the enzyme, dimers with subunits in the range 50 000-60 000 daltons. Partial trypsinolysis mimics this degradation, which is therefore thought to occur through proteolysis. 相似文献
19.
M P Walsh 《Biochemistry》1985,24(14):3724-3730
Myosin light chain kinase plays a central role in the regulation of smooth muscle contraction. The activity of this enzyme is controlled by protein-protein interaction (the Ca2+-dependent binding of calmodulin) and by phosphorylation catalyzed by cAMP-dependent protein kinase. The effects of these two regulatory mechanisms on the conformation of myosin light chain kinase and the locations of the phosphorylation sites, the calmodulin-binding site, and the active site have been probed by limited proteolysis. Phosphorylated and nonphosphorylated myosin light chain kinases were subjected to limited digestion by four proteases having different peptide bond specificities (trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and thrombin), both in the presence and in the absence of bound calmodulin. The digests were compared in terms of gel electrophoretic pattern, distribution of phosphorylation sites, and Ca2+ dependence of kinase activity. A 24 500-dalton chymotryptic peptide containing both sites of phosphorylation was purified and tentatively identified as the amino-terminal peptide. The following conclusions can be drawn: neither phosphorylation nor calmodulin binding induces dramatic changes in the conformation of the kinase; the kinase contains two regions that are particularly susceptible to proteolytic cleavage, one located approximately 25 000 daltons from the amino terminus and the other near the center of the molecule; the two phosphorylation sites are located within 24 500 (probably 17 500) daltons of the amino terminus; the active site is located close to the center of the molecule; the calmodulin-binding site is located in the amino-terminal half of the molecule, between the sites of phosphorylation and the active site, and this region is very susceptible to cleavage by trypsin. 相似文献
20.
Arginine vasopressin (AVP), a nine-amino acid neurohypophyseal hormone, is capable of replacing the helper cell requirement for IFN-gamma production by Lyt-2+ mouse splenic lymphocytes. We present data here showing that the AVP helper signal occurs via interaction with a novel R on splenic lymphocytes and involves primarily the N-terminal six-amino acid cyclic ring (pressinoic acid) with the C-terminal three-amino acid end of AVP playing a minor role. Pressinoic acid was capable of providing help at concentrations similar to those of AVP, whereas oxytocin and isoleucine pressinoic acid were 10- and 100-fold less effective, respectively. Isoleucine pressinoic acid has the same structure as pressinoic acid except for the substitution of isoleucine for phenylalanine in position 3 of the sequence. Consistent with the function data, R binding competitions with splenic lymphocyte membrane preparations showed that AVP and pressinoic acid competed similarly with [3H]AVP, whereas oxytocin and isoleucine pressinoic acid were much less effective competitors. Further characterization of the AVP lymphocyte R was performed using AVP analogues having well defined agonist and antagonist activities on either V1 (vasopressor) R or V2 (antidiuretic) R. The AVP helper signal was blocked by the V1 antagonist [d(CH2)1(5) Tyr(methyl)]AVP but not by another V1 antagonist, [d(CH2)1(5)D-Tyr(ethyl)2Val4]AVP. Both V1-R antagonists were able to block [3H]AVP binding to the V1-R on liver cells, whereas only the V1 antagonist that blocked AVP help was able to compete effectively for the spleen AVP-R. Neither a V2 agonist nor a V2 antagonist had any effect on AVP help in IFN-gamma production. These data strongly indicate the presence of a novel AVP-R on spleen lymphocytes, which is related to the classic V1-R on liver cell membranes. 相似文献