共查询到20条相似文献,搜索用时 15 毫秒
1.
H Brzeska J Szynkiewicz W Drabikowski 《Biochemical and biophysical research communications》1983,115(1):87-93
The exposure of hydrophobic sites on calmodulin, skeletal muscle troponin C and their tryptic fragments was investigated using Phenyl-Sepharose chromatography. A strong binding of both proteins and their fragments corresponding to the NH2-terminal halves of polypeptide chain of respective proteins in the presence of calcium ions was observed. Only a weak interaction with Phenyl-Sepharose or its lack was observed under these conditions for fragments corresponding to the COOH-terminal halves of calmodulin and troponin C, respectively. The elution of the samples from Phenyl-Sepharose column with ethylene glycol gradient allowed to compare relative hydrophobicity of both proteins and their fragments. The results show that hydrophobic properties of calmodulin and troponin C are virtually preserved in their fragments obtained as a result of their cleavage by trypsin in half. They also indicated that the exposure of hydrophobic residues caused by the binding of calcium ions takes place mainly in the NH2-terminal halves of polypeptide chains of both proteins. A simple method of purification of tryptic fragments of both proteins based on the difference in the strength of their interactions with Phenyl-Sepharose is described. 相似文献
2.
S A Berkowitz 《Analytical biochemistry》1987,164(1):254-260
Rapid preparative scale purification of calmodulin from crude bovine brain extract is achieved in a single chromatographic run by physically coupling two different liquid chromatography columns which employ different separation mechanisms. In this case columns packed with newly commercialized 40-microns silica-based hydrophobic interaction and 5-microns micron silica-based weak anion-exchange chromatography media were used. The only sample preparation required for conducting this purification procedure is the addition of salt to the crude brain supernatant to promote the initial binding of calmodulin to the hydrophobic interaction chromatography media. Chromatography carried out on such linear arrangements of columns has been referred to as linear multidimensional liquid chromatography. 相似文献
3.
Calmodulin was isolated and purified from shrimp abdominal muscle by heat precipitation, ion exchange and hydrophobic interaction chromatography. The purified calmodulin was homogeneous when evaluated by polyacrylamide gel electrophoresis. A still remaining contaminant was eliminated by high performance liquid chromatography on a phenyl column. The biological and physicochemical properties of shrimp calmodulin such as amino acid composition, molecular weight and the ability to activate calmodulin-deficient bovine heart phosphodiesterase were compared to those of other invertebrate calmodulins. 相似文献
4.
Native calmodulin binds four calcium ions per molecule and exhibits strong Ca2+-dependent binding to phenyl-Sepharose. In contrast, calmodulin inactivated by oxidation of methionine residues or by deamidation binds fewer calcium ions (two per molecule) and shows relatively weak interaction with phenyl-Sepharose. Calmodulin inactivated by modification of lysine residues still is able to bind four calcium ions per molecule and shows strong binding to phenyl-Sepharose similar to native calmodulin. The results suggest that complete exposure of calmodulin's hydrophobic region occurs only after the binding of four ions of calcium to the calmodulin molecule. Thus, phenyl-Sepharose hydrophobic interaction chromatography might be used to separate active calmodulin from inactive forms of calmodulin obtained by oxidation or heat treatment for prolonged periods. As an example, phenyl-Sepharose chromatography can be used to separate free iodide and inactivated species of calmodulin readily from the active, iodinated form of calmodulin following iodination. 相似文献
5.
Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin. 相似文献
6.
Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions. 相似文献
7.
Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells 总被引:1,自引:0,他引:1
R Wetzker F D B?hmer R Klinger E Müller H Hegewald M Scheven R Grosse 《Biochimica et biophysica acta》1986,854(1):117-123
Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin. 相似文献
8.
Tobias Hahn Nora Geng Katerina Petrushevska-Seebach Michael E. Dolan Marcus Scheindel Pia Graf Kosuke Takenaka Kyo Izumida Lijuan Li Zijian Ma Norbert Schuelke 《Biotechnology progress》2023,39(2):e3316
Mixed-mode chromatography combines features of ion-exchange chromatography and hydrophobic interaction chromatography and is increasingly used in antibody purification. As a replacement for flow-through operations on traditional unmixed resins or as a pH-controlled bind-and-elute step, the use of both interaction modes promises a better removal of product-specific impurities. However, the combination of the functionalities makes industrial process development significantly more complex, in particular the identification of the often small elution window that delivers the desired selectivity. Mechanistic modeling has proven that even difficult separation problems can be solved in a computer-optimized manner once the process dynamics have been modeled. The adsorption models described in the literature are also very complex, which makes model calibration difficult. In this work, we approach this problem with a newly constructed model that describes the adsorber saturation with the help of the surface coverage function of the colloidal particle adsorption model for ion-exchange chromatography. In a case study, a model for a pH-controlled antibody polishing step was created from six experiments. The behavior of fragments, aggregates, and host cell proteins was described with the help of offline analysis. After in silico optimization, a validation experiment confirmed an improved process performance in comparison to the historical process set point. In addition to these good results, the work also shows that the high dynamics of mixed-mode chromatography can produce unexpected results if process parameters deviate too far from tried and tested conditions. 相似文献
9.
Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatrography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin. 相似文献
10.
包涵体蛋白的分离和色谱法体外复性纯化研究进展 总被引:2,自引:0,他引:2
重组蛋白在大肠杆菌中表达多为无活性的包涵体形式,须经洗涤、溶解、复性后才能得到生物活性蛋白。综述了近年来包涵体蛋白分离纯化和复性技术研究进展,重点讨论了色谱法复性技术的应用,包括尺寸排阻色谱、亲和色谱、离子交换色谱、疏水相互作用色谱、固定化脂质体色谱、扩张床吸附色谱的进展情况。 相似文献
11.
Betaine aldehyde dehydrogenase from spinach leaves: purification, in vitro translation of the mRNA, and regulation by salinity 总被引:16,自引:0,他引:16
Spinach (Spinacia oleracea L.) leaves contain a nuclear-encoded chloroplastic betaine aldehyde dehydrogenase (EC 1.2.1.8) which is induced several-fold by salinization. Betaine aldehyde dehydrogenase was purified 2400-fold to homogeneity with an overall yield of 14%. The procedure included fractional precipitation with ammonium sulfate, followed by ion-exchange, hydrophobic interaction, and hydroxyapatite chromatography in open columns, and ion-exchange and hydrophobic interaction chromatography in a fast-protein liquid chromatography system. The betaine aldehyde dehydrogenase had a pI of 5.65, and a broad pH optimum between 7.5 and 9.5. The Km values for NAD+ and NADP+ were 20 and 320 microM, respectively; the Vmax of the reaction with NADP+ was 75% of that with NAD+. The native enzyme is a dimer with subunits of Mr 63,000. Highly specific antiserum was raised against the native enzyme, and was used in conjunction with cell-free translation of leaf poly(A)+ RNA to show (a) that betaine aldehyde dehydrogenase is synthesized as a precursor of Mr 1200 higher than the mature polypeptide, and (b) that both chronic salt stress and salt shock provoke a several-fold increase in the level of translatable message for the enzyme. 相似文献
12.
Katharine Y. Ku Fred R. Butcher 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,631(1):70-78
Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin. 相似文献
13.
Aaron L. Berkovsky Pavel P. Potapov 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,692(2):273
Placental protein 14 was isolated from the biological material of patients undergoing legal abortions. The major part of ballast protein was removed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose. Albumin was separated by chromatography on Blue-Sepharose. Complete purification was obtained by metal-chelate affinity chromatography on Nickel-Chelate Sepharose and hydrophobic interaction chromatography on Phenyl-Sepharose and Octyl-Sepharose. The protein was not exposed to denaturing agents or extreme pH. 相似文献
14.
Direct detection of proteins in high-performance liquid chromatography electrochemistry (LCEC) is difficult. By using on-line, postcolumn photolysis, proteins now can be detected by LCEC at microgram per milliliter levels. The compatibilities of size exclusion chromatography (SEC), reversed-phase chromatography (RPC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC) with photolysis-electrochemical detection is described for proteins together with the analytical figures of merit. Inherent from the advantages of electrochemical detection, the method is sensitive and selective. 相似文献
15.
Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography 总被引:45,自引:0,他引:45
R Gopalakrishna W B Anderson 《Biochemical and biophysical research communications》1982,104(2):830-836
Calmodulin binds quantitatively to phenyl-Sepharose and octyl-Sepharose affinity columns in the presence of micromolar concentrations of Ca2+. In addition to EGTA, calmodulin also can be eluted from these affinity columns with low ionic strength buffer, non-ionic detergent (i.e., 1% Triton X-100), or ethylene glycol (50%), suggesting hydrophobic interaction. Using hydrophobic interaction chromatography calmodulin can be purified to homogeneity from bovine brain homogenate in a single step. For large-scale purification the protein fraction containing calmodulin was concentrated by isoelectric precipitation prior to application to the affinity column. The yield obtained by this procedure (160–180 mg calmodulin per kg brain) is significantly greater, and the time required (~ 5 hr) is substantially less, than that of previously described procedures for calmodulin purification. It is apparent that phenyl-Sepharose offers several advantages over phenothiazine-Sepharose for affinity purification of calmodulin. 相似文献
16.
Phosphatidylinositol-specific phospholipase C from Bacillus cereus: improved purification, amino acid composition, and amino-terminal sequence 总被引:3,自引:0,他引:3
J J Volwerk P B Wetherwax L M Evans A Kuppe O H Griffith 《Journal of cellular biochemistry》1989,39(3):315-325
Phosphatidylinositol-specific phospholipase C was purified in a 27% yield from the culture medium of Bacillus cereus by a combination of ammonium sulfate precipitation and ion-exchange and hydrophobic interaction chromatography. The purified enzyme was free of other phospholipase C-type activities and exhibited a high specific activity of approximately 1,300 units/mg. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular weight of about 35 kDa. The sequence of the first 29 N-terminal amino acids was also determined. 相似文献
17.
Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin 总被引:5,自引:0,他引:5
Highly purified tryptic peptides of calmodulin have been obtained by high-performance liquid chromatography. Tryptic cleavage of calmodulin in the presence of Ca2+ results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-148. In the absence of Ca2+, trypsin cleavage yields fragments 1-106, 1-90, and 107-148. Only fragments 78-148 and 1-106 are still able to stimulate the purified Ca2+-ATPase of erythrocytes, albeit much less efficiently on a molar basis, than intact calmodulin. On the other hand, the same fragments were unable to stimulate the calmodulin-dependent cyclic nucleotide phosphodiesterase, even at 1000-fold molar excess (shown also by Newton, D.L., Oldewurtel, M.D., Krinks, M.H., Shiloach, J., and Klee, C.B. (1984) J. Biol. Chem. 259, 4419-4426). This points to the importance of the carboxyl-terminal half of calmodulin and especially of Ca2+-binding region III in the interaction of calmodulin with the Ca2+-ATPase and provides clear evidence that calmodulin interacts differently with different targets. Oxidation of methionine(s) of fragment 78-148 with N-chlorosuccinimide removes the ability of this fragment to stimulate the ATPase. 相似文献
18.
Hydrophobic-ionic chromatography: its application to microbial glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase 总被引:1,自引:0,他引:1
I Sasaki H Gotoh R Yamamoto H Tanaka K Takami K Yamashita J Yamashita T Horio 《Journal of biochemistry》1982,91(5):1555-1561
Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH less than or equal to 4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobic-ionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH. 相似文献
19.
In this study we describe the identification of four soluble forms of cyclic nucleotide phosphodiesterase from chicken gizzard smooth muscle. These isoenzymes were separated from one another by ion-exchange chromatography on DEAE-cellulose and by calmodulin-Sepharose affinity chromatography. Each form migrates as a single discrete band when it is electrophoresed on non-denaturing polyacrylamide gels and stained for phosphodiesterase activity. Each form is also eluted as a single peak on gel-permeation chromatography, giving apparent Mr values of 114 000, 116 000, 122 000 and 59 000. All four enzymes have apparent Km values in the 0-20 microM range, although their relative specificities for cyclic AMP and cyclic GMP differ. Two of the forms bind to calmodulin in a Ca2+-dependent manner; however, only one is activated by calmodulin. The interaction of the second calmodulin-binding form with calmodulin is disrupted by the papaverine derivative verapamil without significantly altering the hydrolytic activity of the enzyme. 相似文献
20.
Characterization of the calmodulin-binding sites of muscle phosphofructokinase and comparison with known calmodulin-binding domains 总被引:7,自引:0,他引:7
Calmodulin has been shown to interact with high affinity with muscle phosphofructokinase (Mayr, G. W. (1984) Eur. J. Biochem. 143, 513-520, 521-529). In this study, direct binding measurements indicated that each of the two subunits of dimeric phosphofructokinase bound two calmodulins with Kd values of about 3 nM and 1 microM, respectively, in a strictly Ca2+-dependent way. To get more detailed information about this interaction, calmodulin-binding fragments were isolated from a CNBr digest of phosphofructokinase using affinity chromatography on calmodulin-agarose. Two fragments, M11 (Mr 3080) and M22 (Mr 8060), formed a 1:1 stoichiometric complex with Ca2+-calmodulin. The amino acid sequences of these fragments were determined, and their positions in the three-dimensional structure-model of phosphofructokinase are proposed. Fragment M11, which binds to calmodulin with the higher affinity (Kd 11.4 nM), is located in a region of the subunit where two dimers have been proposed to make contacts if associating to active tetrameric enzyme. A stabilization of the dimeric form of the enzyme by binding of calmodulin supports this location of M11. The weaker binding fragment M22 (Kd 198 nM) corresponds to the C-terminal part of the polypeptide and contains the site which is phosphorylated by cAMP-dependent protein kinase. Both fragments have structural properties in common with the isolated calmodulin-binding domains of myosin light chain kinase: two cationic segments rich in hydrophobic residues, one constantly possessing a tryptophan, and the other exhibiting an amino acid sequence resembling sites phosphorylated by cAMP-dependent protein kinase. 相似文献