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Preparative biochemistry最新文献

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Purification of porcine heart latent protein phosphatase Fc.M. 猪心脏潜伏蛋白磷酸酶的纯化。
Preparative biochemistry Pub Date : 1992-09-01 DOI: 10.1080/10826069208021371
M D Schuchard, S D Killilea
{"title":"Purification of porcine heart latent protein phosphatase Fc.M.","authors":"M D Schuchard,&nbsp;S D Killilea","doi":"10.1080/10826069208021371","DOIUrl":"https://doi.org/10.1080/10826069208021371","url":null,"abstract":"<p><p>Latent protein phosphatase, Fc.M, was purified from porcine heart extracts by a procedure involving precipitation at pH 5.0, DEAE-Sephacel chromatography, ammonium sulfate fractionation, chromatography on phenyl-Sepharose, Biogel-A 0.5m and poly-L-lysine-agarose. The purified enzyme had a specific activity of 12,200 nanomoles of phosphate released from phosphorylase a/mg protein when assayed following activation by pretreatment with Mn++ and trypsin in the presence of 0.2 M NaCl. The enzyme is a heterodimer of 66 kDa composed of a catalytic (37 kDa) and a modulator (31 kDa) subunit.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 3-4","pages":"199-213"},"PeriodicalIF":0.0,"publicationDate":"1992-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021371","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12511446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Purification and characterization of porcine heart type 2A protein phosphatases. 猪心脏2A型蛋白磷酸酶的纯化与鉴定。
Preparative biochemistry Pub Date : 1992-09-01 DOI: 10.1080/10826069208021375
A K Erickson, S D Killilea
{"title":"Purification and characterization of porcine heart type 2A protein phosphatases.","authors":"A K Erickson,&nbsp;S D Killilea","doi":"10.1080/10826069208021375","DOIUrl":"https://doi.org/10.1080/10826069208021375","url":null,"abstract":"<p><p>Protein phosphatases 2A1 and 2A2 were isolated from porcine heart tissue extracts by precipitation at pH 5.0 and separated by chromatography on DEAE-Sephacel. Phosphatase 2A1 was then purified to apparent homogeneity by chromatography on phenyl-Sepharose, aminohexyl-Sepharose, Sephacryl S-300, and L-tyrosine-agarose. Phosphatase 2A2 was purified to apparent homogeneity by chromatography on phenyl-Sepharose, DEAE-Sephacel, aminohexyl-Sepharose and L-tyrosine-agarose. Purified phosphatases 2A1 and 2A2 had specific activities of 2200 and 2710 nanomoles of phosphate released from phosphorylase a/mg protein, respectively. The apparent molecular weights of phosphatases 2A1 and 2A2 on gel filtration were 155 and 105 kDa, respectively. Both enzymes contain 70 and 37 kDa subunits and 2A1 also contains a 57 kDa subunit. The 37 kDa catalytic subunit (2Ac) was obtained from the purified phosphatases by treatment with room temperature ethanol followed by sucrose density gradient centrifugation or gel filtration chromatography.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 3-4","pages":"257-74"},"PeriodicalIF":0.0,"publicationDate":"1992-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021375","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12511447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Purification of rat urinary kallikrein: comparative studies with rat submandibular gland kallikrein-like serine protease. 大鼠尿钾化肽的纯化:与大鼠颌下腺钾化肽样丝氨酸蛋白酶的比较研究。
Preparative biochemistry Pub Date : 1992-09-01 DOI: 10.1080/10826069208021374
G S Bedi
{"title":"Purification of rat urinary kallikrein: comparative studies with rat submandibular gland kallikrein-like serine protease.","authors":"G S Bedi","doi":"10.1080/10826069208021374","DOIUrl":"https://doi.org/10.1080/10826069208021374","url":null,"abstract":"<p><p>A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 3-4","pages":"239-56"},"PeriodicalIF":0.0,"publicationDate":"1992-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021374","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12462445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 5
Preparation of ferric adsorbent paper and its interaction with phosphate-containing biomolecules. 铁吸附纸的制备及其与含磷生物分子的相互作用。
Preparative biochemistry Pub Date : 1992-09-01 DOI: 10.1080/10826069208021370
R Toomik, P Toomik
{"title":"Preparation of ferric adsorbent paper and its interaction with phosphate-containing biomolecules.","authors":"R Toomik,&nbsp;P Toomik","doi":"10.1080/10826069208021370","DOIUrl":"https://doi.org/10.1080/10826069208021370","url":null,"abstract":"<p><p>A procedure for the synthesis of a selective adsorbent for phosphate-containing biomolecules is described. The sorbent is based on Whatman chromatography paper, which is activated with epichlorohydrine, followed by the coupling of iminodiacetic acid to the active surface of the sorbent. The immobilized complex-forming chelating groups are saturated with ferric ions. The synthesized adsorbent is a counterpart to Chelating Sepharose and makes it possible to extend the use of immobilized ferric chelating groups for analytical purposes. It displays a high affinity towards compounds containing free terminal phosphate groups (phosphopeptides, nucleotides). The results of the binding experiments are compared to the corresponding data obtained with Chelating Sepharose gels.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 3-4","pages":"183-97"},"PeriodicalIF":0.0,"publicationDate":"1992-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021370","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12462484","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
Isolation, purification and biochemical characterization of human placental interferons by tandem high-performance affinity chromatography. 人胎盘干扰素的分离纯化及串联高效亲和层析的生化特性研究。
Preparative biochemistry Pub Date : 1992-06-01 DOI: 10.1080/10826069208021362
G Aboagye-Mathiesen, F D Tóth, A M Dalsgaard, P M Petersen, V Zachar, P Ebbesen
{"title":"Isolation, purification and biochemical characterization of human placental interferons by tandem high-performance affinity chromatography.","authors":"G Aboagye-Mathiesen,&nbsp;F D Tóth,&nbsp;A M Dalsgaard,&nbsp;P M Petersen,&nbsp;V Zachar,&nbsp;P Ebbesen","doi":"10.1080/10826069208021362","DOIUrl":"https://doi.org/10.1080/10826069208021362","url":null,"abstract":"<p><p>Human placental trophoblasts, fibroblasts and the trophoblast-derived malignant cell JAR are potent producers of interferons (IFNs) when stimulated with Sendai virus. The three cell lines produced different levels and compositions of IFN-alpha subtypes and IFN-beta. Anti-IFN globulins, Cibacron Blue F3GA and Concanavalin A were covalently immobilized on pressure-stable, macroporous polymeric matrices derivatized with vinyl sulphone (HEMA-BIO 1000 VS and HEMA 1000 VS). These supports were packed in biocompatible PEEK columns and were coupled with switching valves, to develop a tandem high-performance affinity chromatographic (HPAC) method for the isolation, purification and biochemical characterization of the IFNs produced in Sendai virus-stimulated human placental trophoblasts, fibroblasts and trophoblast-derived malignant cell, JAR, cultures. Silver-stained SDS-PAGE and gel densitometric analysis revealed the purity of the purified proteins to be between 94 and 98%. Specific activities of the purified IFNs ranged between 0.37-2.76 x 10(8) IU/mg of protein with cumulative recoveries between 90 and 92.2%. The purified IFN components exhibited quantitatively different antiviral activities in human and bovine cell lines. The utility of the tandem method for the purification and characterization of human type 1 IFNs produced from other cell lines are also discussed.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 2","pages":"105-21"},"PeriodicalIF":0.0,"publicationDate":"1992-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021362","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12550641","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 10
Gram scale purification and preparation of rabbit liver zinc metallothionein. 兔肝锌金属硫蛋白的克级纯化及制备。
Preparative biochemistry Pub Date : 1992-06-01 DOI: 10.1080/10826069208021365
R D Comeau, K W McDonald, G L Tolman, M Vasak, F A Liberatore
{"title":"Gram scale purification and preparation of rabbit liver zinc metallothionein.","authors":"R D Comeau,&nbsp;K W McDonald,&nbsp;G L Tolman,&nbsp;M Vasak,&nbsp;F A Liberatore","doi":"10.1080/10826069208021365","DOIUrl":"https://doi.org/10.1080/10826069208021365","url":null,"abstract":"<p><p>A simplified procedure for purifying gram quantities of rabbit liver metallothionein (MT) using gel filtration and anion exchange chromatography is presented. The MT purification made use of anion exchange batch elution chromatography which greatly shortened the procedure. Quantitation techniques for use with crude and purified MT are discussed. This paper also describes the preparation of large amounts of ZnMT from Cd,ZnMT.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 2","pages":"151-64"},"PeriodicalIF":0.0,"publicationDate":"1992-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021365","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12464438","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 26
Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to protein A Sepharose 4 Fast Flow. 温度、流速及结合缓冲液组成对小鼠单克隆IgG1抗体对蛋白A Sepharose 4快速流动吸附的影响
Preparative biochemistry Pub Date : 1992-06-01 DOI: 10.1080/10826069208021364
A P van Sommeren, P A Machielsen, T C Gribnau
{"title":"Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to protein A Sepharose 4 Fast Flow.","authors":"A P van Sommeren,&nbsp;P A Machielsen,&nbsp;T C Gribnau","doi":"10.1080/10826069208021364","DOIUrl":"https://doi.org/10.1080/10826069208021364","url":null,"abstract":"<p><p>The binding capacity of protein A Sepharose 4 Fast Flow for mouse IgG1 monoclonal antibodies (mabs) appears to be highly dependent on the buffer composition with respect to both concentration and ion type. Depending on the particular mab dynamic binding capacities up to 20 mg mab per ml gel could be obtained, when these mabs were isolated from supernatants of protein free hollow fibre cell culture systems. Variation of linear flow rate from 10 up to 300 cm/h and temperature (4 degrees C versus 25 degrees C) had a slight effect on the dynamic binding capacity, when a high ionic strength buffer was used during adsorption. Applying optimum binding conditions, final IgG fractions with a purity of more than 95% monomeric IgG were obtained. However, as side effect of the use of binding buffers with high ionic strength, the binding of acid proteases was also promoted.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 2","pages":"135-49"},"PeriodicalIF":0.0,"publicationDate":"1992-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021364","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12787440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 17
Isolation and purification of extracellular matrix vesicles from blood vessels. 血管细胞外基质囊泡的分离纯化。
Preparative biochemistry Pub Date : 1992-06-01 DOI: 10.1080/10826069208021361
B J Martin, S Thomas, N S Greenhill, P A Ryan, P F Davis, W E Stehbens
{"title":"Isolation and purification of extracellular matrix vesicles from blood vessels.","authors":"B J Martin,&nbsp;S Thomas,&nbsp;N S Greenhill,&nbsp;P A Ryan,&nbsp;P F Davis,&nbsp;W E Stehbens","doi":"10.1080/10826069208021361","DOIUrl":"https://doi.org/10.1080/10826069208021361","url":null,"abstract":"<p><p>Extracellular membrane-bound vesicles (called matrix vesicles) which occur in abundance in atherosclerotic blood vessels are believed to be associated with lipid accumulation and calcification. A technique has been developed to isolate them from experimental aneurysms in sheep in which they are known to be plentiful. The matrix vesicles were isolated by differential centrifugation following extraction by hypotonic salt solution. Most of the vesicles were pelleted at 30,000g and fell within the size range of matrix vesicles in situ in the aneurysmal wall. Preliminary characterization of the enzymatic activities indicates that many of these vesicles are formed from cell membranes rather than being derived from lysosomes, mitochondria or endoplasmic reticulum. Morphologically they are similar to matrix vesicles of other mineralizing tissues.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 2","pages":"87-103"},"PeriodicalIF":0.0,"publicationDate":"1992-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021361","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12787442","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Isolation and purification of the extracellular and intracellular portions of the beta subunit of (Na+,K+)-ATPase. (Na+，K+)- atp酶β亚基细胞外和细胞内部分的分离和纯化。
Preparative biochemistry Pub Date : 1992-06-01 DOI: 10.1080/10826069208021363
A Ataei, E T Wallick
{"title":"Isolation and purification of the extracellular and intracellular portions of the beta subunit of (Na+,K+)-ATPase.","authors":"A Ataei,&nbsp;E T Wallick","doi":"10.1080/10826069208021363","DOIUrl":"https://doi.org/10.1080/10826069208021363","url":null,"abstract":"<p><p>The beta subunit of lamb kidney (Na+,K+)-ATPase was isolated by size exclusion high performance liquid chromatography. Treatment of the beta subunit with formic acid yielded two peptide fragments which were purified via reversed phase high performance liquid chromatography. These peptides were identified by sodium dodecylsulfate polyacrylamide gel electrophoresis, amino acid analysis and N-terminal sequencing as (Pro 94-Ser 302), a largely hydrophilic peptide which comprises the major portion of the extracellular domain including six Cys residues which participate in disulfide bond formation and three glycosylation sites and a smaller peptide (Ala 1-Asp 93) which contains the single membrane spanning region and the intracellular domain.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 2","pages":"123-33"},"PeriodicalIF":0.0,"publicationDate":"1992-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021363","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12494757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Two methods to avoid the effect of endogenous protein inhibitors during the assay of protein kinase C activity in tissue extracts. 两种方法避免内源性蛋白抑制剂对组织提取物中蛋白激酶C活性的影响。
Preparative biochemistry Pub Date : 1992-06-01 DOI: 10.1080/10826069208021366
P Ekman, M Eller, U Ragnarsson, L Engström
{"title":"Two methods to avoid the effect of endogenous protein inhibitors during the assay of protein kinase C activity in tissue extracts.","authors":"P Ekman,&nbsp;M Eller,&nbsp;U Ragnarsson,&nbsp;L Engström","doi":"10.1080/10826069208021366","DOIUrl":"https://doi.org/10.1080/10826069208021366","url":null,"abstract":"<p><p>Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.</p>","PeriodicalId":20391,"journal":{"name":"Preparative biochemistry","volume":"22 2","pages":"165-75"},"PeriodicalIF":0.0,"publicationDate":"1992-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826069208021366","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12787441","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 5
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