Facile analysis and purification of deblocked N-terminal pyroglutamyl peptides with a strong cation-exchange sulfoethyl aspartamide column

A high-performance strong cation-exchange Sulfoethyl Aspartamide column was used to analyze and purify five N-terminal pyroglutamyl peptides after treatment with Pyroglutamate Aminopeptidase. The resulting deblocked N-1 peptides possess an increased positive charge and are therefore retained to a greater extent by the column. Salt gradient elution in a pH 3 mobile phase was then used to recover the desired peptides and the purified deblocked peptides were directly subjected to N-terminal sequence analysis. The same digests were also chromatographed on a C18 reversed-phase column using standard trifluoroacetic acid-acetonitrile gradient elution. The elution order for the parent peptide and the N-1 peptide on the reversed-phase column was reversed from that on the Sulfoethyl Aspartamide column and the resolution of the two peptides obtained on the reversed-phase column was less than that observed on the cation-exchange column. In addition, the Sulfoethyl Aspartamide column was shown to be useful to monitor the extent of N-terminal glutamine cyclization formed during peptide purification and storage.     

Phosphorylation of chondroitin sulfate in proteoglycans from the swarm rat chondrosarcoma

Proteoglycans isolated from the Swarm rat chondrosarcoma were shown to contain 35 mol of phosphate/mol of proteoglycan. While 20% of this phosphate was released by digestion with dilute alkali in the presence of sodium borohydride and is presumably of the phosphoserine/phosphothreonine type, 78% of the phosphate copurified with the peptide-free chondroitin sulfate chains. When chondroitin sulfate chains purified by ethanol precipitation or Sephacryl S200 column chromatography were digested with chondroitinase AC and the digests chromatographed on Bio-Gel P-4, the phosphate co-migrated with a carbohydrate fragment that contained 2 glucuronic acid (one as delta 4,5-unsaturated sugar), 1-galactosamine, 2-galactose, and 1-phosphate residue/xylitol. A second fragment of similar composition but lacking phosphate was also recovered in a ratio of about 3 to 1 relative to the phosphorylated fragment. The phosphate in the chondroitin sulfate linkage region fragment had the alkaline phosphatase sensitivity as well as 31P NMR spectra of a monophosphate esterified to a secondary sugar alcohol. The phosphate was localized on the C-2 of the chain initiating xylose since these residues as xylitol showed a delayed release during acid hydrolysis and the xylitol was recovered intact after periodate oxidation. In the chondrosarcoma, 2-phosphoxylose appears to be a normal synthetic product since [32P]phosphate was readily incorporated into the proteoglycan and the incorporated isotope had similar biochemical properties as the unlabeled phosphate.     

Autophosphorylation of Mucor rouxii cAMP-dependent protein kinase and its role in holoenzyme activation.

Two phosphoproteins of 53,000 and 63,000 mol. wt detected in partially purified preparations of Mucor rouxii cAMP-dependent protein kinase submitted to phosphorylation conditions with [gamma-32P]ATP are demonstrated to be the result of the autophosphorylation of its regulatory subunit, according to the following criteria: (1) linearity of phosphate incorporation with enzyme sample; (2) independence of phosphate incorporation on temperature; (3) correlation of the phosphoproteins with enzymatic activity in a DEAE-Sepharose chromatography; (4) specific elution of the phosphorylated proteins from cAMP-agarose; (5) phosphorylation of the purified regulatory subunit. Antibodies specific against Mucor regulatory subunit detected an intact subunit of 72,000 mol. wt in crude extracts. Autophosphorylation of the fungal protein kinase A promotes activation of the holoenzyme by cAMP since: (1) under conditions of partial activation, increase of activity is observed when using the phosphoform of the enzyme; (2) release of free catalytic subunit from cAMP-agarose is enhanced when the holoenzyme is previously phosphorylated.     

Purification to homogeneity of extracellular polygalacturonase and isoenzymes of pectate lyase of Erwinia carotovora subsp. atroseptica by column chromatography

Extracellular polygalacturonase (PG) and two pectate lyase isoenzymes (PLI and PLII) produced by a 48 h culture of Erwinia carotovora subsp. atroseptica in pectate-based medium were purified 2027, 2036 and 2374-fold respectively to homogeneity with corresponding 59%, 61% and 32% recovery. This was achieved first by ion exchange chromatography on a S-Sepharose fast flow column with 20 mmol/1 Tris at pH 8.0 followed by elution of bound proteins with a 1 mol/1 NaCl gradient which separated PG from PL. The two enzymes were then further purified to homogeneity (assessed by SDS-PAGE) by selective adsorption chromatography on a hydroxyapatite column equilibrated with distilled water; PG was eluted with a 3 mol/1 KCl gradient and PLI with a 3 mol/1 KCl gradient followed by a 1.2 mol/1 PO₄ buffer pH 6.5 gradient to elute PLII. The Mr of the three enzymes determined by SDS-PAGE was 39 kDa and the pI values for PG, PLI and PII were 10.3, 10.3 and 10.0 respectively as determined by isoelectric focusing (IEF)-gel electrophoresis followed by activity staining.     

Phosphorylation of chicken cardiac C-protein by calcium/calmodulin-dependent protein kinase II.

Chicken cardiac C-protein was readily phosphorylated by purified calcium/calmodulin-dependent protein kinase II (CaM-kinase II). Maximum incorporation was about 4 mol of 32P/mol of C-protein subunit. Peptide mapping indicated that some of the sites phosphorylated by CaM-kinase II were located on the same phosphopeptides obtained when C-protein was phosphorylated by the cAMP-dependent protein kinase (peptides T1, T2, and T3). There was a fourth peptide (T3a) which was unique to CaM-kinase II phosphorylation. 32P-Amino acid analysis showed that essentially all of the 32P of peptides T1, T2, and T3a was in phosphoserine. cAMP-dependent protein kinase incorporated 32P only into threonine of peptide T3. Threonine was the preferred site of phosphorylation by CaM-kinase II, but there was significant phosphorylation of a serine in peptide T3. Partially purified C-protein preparations contained an associated calcium/calmodulin-dependent protein kinase. Peptide maps obtained from C-protein phosphorylated by the endogenous kinase were similar to those obtained from C-protein phosphorylated by CaM-kinase II. However, the ratio of phosphothreonine to phosphoserine in peptide T3 was lower. This was due to a contaminating phosphatase in the partially purified C-protein which preferentially dephosphorylated the phosphothreonine of peptide T3. It is suggested that the calcium/calmodulin-dependent protein kinase associated with C-protein is similar or identical to CaM-kinase II and that CaM-kinase II may play a role in the phosphorylation of C-protein in the heart.     

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.     

Principal neurofilament-associated protein kinase in squid axoplasm is related to casein kinase I

A cytoskeletal extract of pure axoplasm, highly enriched with neurofilaments (ANF), was prepared from the giant axon of the squid. This ANF preparation also contained potent kinase activities which phosphorylated the Mr greater than 400,000 (high molecular weight) and Mr 220,000 squid neurofilament protein subunits. High salt (1 M) extraction of this ANF preparation solubilized most of the neurofilament proteins and kinase activities and gel filtration on an AcA 44 column separated these two components. The neurofilaments eluted in the void volume of the column while the kinase activities eluted in the 17-44-kDa range of the column. Two major kinase activities were measured in this peak of activity. One of these strongly phosphorylated the phosphate acceptor peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and was completely inhibited by the selective inhibitor of cAMP-dependent kinase Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- NH2 (Wiptide). Since addition of cAMP did not stimulate activity, this suggested that this kinase was a free catalytic subunit of cAMP-dependent kinase associated with the neurofilaments. The second kinase activity most effectively phosphorylated alpha-casein, and this activity was not affected by Wiptide. The alpha-casein phosphorylating activity (ANF kinase) was the principal activity responsible for neurofilament protein phosphorylation, and was not inhibited by various inhibitors against second messenger regulated kinases, suggesting it was related to the casein kinase family. Four lines of evidence indicate ANF kinase was similar to casein kinase I. These were: 1) the apparent molecular weight determined by gel filtration and the chromatographic elution profile on phosphocellulose column corresponded to casein kinase I; 2) heparin, an inhibitor of casein kinase II at 2-5 micrograms/ml, stimulated both ANF kinase and purified casein kinase I at these concentrations, while CKI-7, a relatively selective inhibitor of casein kinase I, inhibited ANF kinase in a comparable dose-response fashion; 3) purified casein kinase I strongly phosphorylated both ANF protein subunits (like ANF kinase) whereas casein kinase II was relatively ineffective; and 4) tryptic peptide maps of the HMW and Mr 220,000 neurofilament proteins after phosphorylation by ANF kinase or purified casein kinase I showed similar 32P-peptide patterns.     

Isolation of a catalytically competent phosphorylated tyrosine kinase from Rous sarcoma virus-induced rat tumor by immunoadsorption to and hapten elution from phosphotyrosine binding antibodies.

A procedure has been developed for the isolation of a catalytically competent phosphorylated tyrosine kinase (RSV Y-kinase) from avian sarcoma virus-induced rat tumors. The procedure involves reaction of partially purified RSV Y-kinase with ATP to effect tyrosyl phosphorylation of catalytically competent RSV Y-kinase. Tyrosyl phosphorylated RSV Y-kinase was isolated from the heterogeneous reaction mixture by immunoadsorption on immobilized phosphotyrosyl binding antibodies and elution with the hapten p-nitrophenyl phosphate. Estimation of the phosphate content of the purified phosphorylated RSV Y-kinase indicated that 1-3 tyrosyl groups had been phosphorylated upon reaction with ATP. The specific activity toward histone 2B of the purified phosphorylated RSV Y-kinase was at least 30-fold greater than that estimated for the RSV Y-kinase prepared previously by immunoadsorption on immobilized antiserum from tumor bearing rabbits.     

Purification and characterization of wheat and pine small basic protein substrates for plant calcium-dependent protein kinase.

A wheat basic protein (WBP) was purified to homogeneity from wheat germ by a protocol involving extraction, centrifugation, batchwise elution from carboxymethylcellulose (CM-52), acidification with trifluoroacetic acid, neutralization and HPLC on a SP5PW cation exchange column. WBP is a 10 kDa protein and is phosphorylated on serine residues by wheat germ Ca(2+)-dependent protein kinase (CDPK). [32P]phosphoWBP exactly comigrates with WBP on SDS-PAGE. WBP does not inhibit either wheat germ CDPK or calmodulin-dependent myosin light chain kinase. Apart from histone H1, WBP is the best endogenous substrate yet found for wheat embryo CDPK. A 12 kDa pine basic protein (PBP) was purified to homogeneity from seeds of stone pine (Pinus pinea L.) by a simple procedure involving batchwise elution from carboxymethylcellulose and cation exchange HPLC. PBP is also a good substrate for CDPK and is phosphorylated on Ser residues. N-terminal sequencing of WBP and PBP revealed that these proteins are homologous to a family of small basic plant proteins having a phospholipid transfer function.     

The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.

Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

Phosphorylation of a single subunit of the epithelial Na+ channel protein following vasopressin treatment of A6 cells

Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.     

Expression of the A subunit of protein phosphatase 2A and characterization of its interactions with the catalytic and regulatory subunits.

The alpha form of the A subunit of human protein phosphatase 2A was expressed in insect cells following infection with a recombinant baculovirus. A alpha was expressed as a soluble protein that comprised approximately 10% of total cellular protein. The expressed A alpha subunit was purified by chromatography on amino-hexyl-Sepharose and Mono Q with a yield of 2 mg/500-ml culture. The recombinant protein had the same apparent molecular mass as the bovine cardiac protein and was devoid of myosin light chain phosphatase activity. Biological activity of expressed A was assessed by assays of complex formation with the catalytic (C) and B subunits, purified from bovine cardiac tissue, and by inhibition of phosphatase activity. Purified A alpha had a high apparent affinity for C (IC50 = 0.10 nM) and bound with a stoichiometry of 1 mol of A/mol of C. Interaction of A alpha with the catalytic subunit caused a maximal inhibition of myosin light chain and phosphorylase phosphatase activities of 50 and 79%, respectively. The AC complex prepared by reconstitution of recombinant A alpha with C had the same electrophoretic mobility in nondenaturing polyacrylamide gels and the same elution volume when chromatographed on a size exclusion column as the native AC complex purified from cardiac muscle. Similar chromatographic profiles were also observed for the heterotrimer reconstituted from recombinant A alpha, purified B and C, and the native bovine cardiac heterotrimeric holoenzyme. Cross-linking of the native enzyme and the reconstituted heterotrimer generated the same pattern of high molecular weight species. Immunological analyses of these complexes demonstrated that distinct cross-linked forms composed of ABC, AC, AB, and BC were obtained. These results suggest that each of the three subunits of protein phosphatase 2A forms direct contacts with both of the others.     

Affinity purification and characterization of myristoylated alanine-rich protein kinase C substrate (MARCKS) from bovine brain. Comparison of the cytoplasmic and the membrane-bound forms.

A major in vivo substrate of Ca(2+)-phospholipid-dependent protein kinase (myristoylated alanine-rich C-kinase substrate (MARCKS)) has been purified to apparent homogeneity from the particulate as well as from the cytoplasmic fractions of calf brain using a calmodulin affinity column. The two preparations were characterized and compared with various biochemical and biophysical techniques. Although they behave similarly in various chromatographic procedures during purification, their elution positions from the gel filtration column are markedly different. Stokes radii of 85 and 45 A were measured for the cytoplasmic and membrane MARCKS, respectively. Once purified, however, they show a similar small Stokes radius (45 A), suggesting the dissociation of a component or a drastic conformational change in the cytoplasmic preparation during purification. The electrospray mass spectroscopic analysis of the two preparations revealed the existence of at least three major subpopulations with molecular mass differences of 80 daltons, which suggests the presence of protein phosphorylated in different degrees. The cytoplasmic preparation contains more phosphorylated species compared with the membrane preparation, whereas the calculated molecular weight of each peak was indistinguishable between the two preparations. Correspondingly, when the two preparations were phosphorylated by purified protein kinase C in vitro, more phosphate groups were transferred to the membrane preparation (4 mol/mol) than to the cytoplasmic preparation (2.9 mol/mol). A significant difference was also observed in the inhibition of calmodulin of the phosphorylation reaction. On the other hand, the circular dichroism of the two preparations showed similar spectra rich in random coil with little contribution of alpha-helix (approximately 10%), suggesting that there is not a significant difference in the overall conformation. These results clearly established that the two preparations are the same protein coded by a single gene but they differ in their degree of phosphorylation, and that the difference observed in their Stokes radius is due to the presence of an unidentified factor that is removed from the cytoplasmic MARCKS during purification.     

糖原合成酶激酶-3对τ蛋白磷酸化作用的研究

糖原合成酶激酶 ３（ Ｇ Ｓ Ｋ ３）在 ３０℃与 τ蛋白保温 ４ ｈ 可催化 １７±０４ ｍ ｏｌ磷酸参入 １ ｍ ｏｌτ蛋白 将磷酸化的 τ蛋白经胰蛋白酶消化, Ｆｅ Ｃｌ３ 亲和柱分离及 Ｃ１８反相高压液相层析纯化后,再用高压电泳,手工 Ｅｄｍ ａｎ 降解及自动氨基酸序列分析等检测技术,对其磷酸化位点进行鉴定 结果发现： Ｇ Ｓ Ｋ ３ 可使 τ蛋白 Ｔｈｒ １８１, Ｓｅｒ １８４, Ｓｅｒ ２６２, Ｓｅｒ ３５６ 和 Ｓｅｒ ４００ 发生磷酸化 其中 Ｓｅｒ ２６２ 和 Ｓｅｒ ４００ 为 Ａｌｚｈｅｉｍ ｅｒ 病（ Ａ Ｄ）τ蛋白的异常磷酸化位点根据上述磷酸化作用仅轻度抑制τ蛋白生物学活性,推测： Ａ Ｄ τ蛋白 Ｓｅｒ ２６２ 和 Ｓｅｒ ４００ 的磷酸化可能不是决定其生物功能的关键性位点,单纯 Ｇ Ｓ Ｋ ３ 不能复制 Ａ Ｄ 样 τ蛋白的病理改变      

Leucyl-transfer ribonucleic acid synthetase in senescing tobacco leaves

1. The activity of leucyl-tRNA synthetase obtained from tobacco leaves declined by 50% over a period of 4 days senescence induced by detachment. In addition tRNA(Leu) from senescing leaves was charged to a lesser extent than tRNA(Leu) extracted from mature leaves immediately after detachment. 2. tRNA(Leu) was charged with a synthetase preparation from either mature or senescent leaves and chromatographed on an RPC 3 column. The elution profile showed that the marked decline in specific activity of leucyl-tRNA synthetase in senescent leaves was not associated with a loss of acylation of any isoacceptor of tRNA(Leu).     

Ion-exchange chromatography of a dinucleotide preparation from controlled alkaline hydrolysis of ribonucleic acids

With the aim of preparing the 16 possible ribodinucleotides derived from the four principal ribonucleotides, we made a kinetic study to determine the optimum conditions for a partial alkaline hydrolysis of RNA. Satisfactory results were obtained when the hydrolysis of RNA (1g.) in 0.2m-sodium hydroxide (100ml.) at 37 degrees was ceased approx. 10min. after the start of the reaction. The relative yields of the monomer, dimer and trimer fractions were approx. 41:30:15 in E(260) units, indicating that the reaction conditions were reasonably close to those required from kinetic considerations. The partial alkaline hydrolysate was chromatographed on a column of DEAE-Sephadex A-25 in the presence of 7m-urea. The dinucleotide fraction thus obtained was subjected to a subsequent chromatography on Dowex 1 (X2) under acidic conditions to separate the mixture according to base composition and base sequence. The results were satisfactory, and most of the 32 dinucleotides [i.e. 16 XpYp(3') plus 16 XpYp(2')] were fractionated by this single chromatographic procedure. The present method should be useful for further study of oligonucleotides as a time-saving method for the simultaneous preparation of a variety of dinucleotides. Further, examination of the present chromatographic pattern has provided several empirical criteria useful for the identification of oligonucleotides other than dimers appearing in the elution profile of Dowex 1 (X2) column chromatography under similar conditions.     

Isolation of a trypsin inhibitor from Echinodorus paniculatus seeds by affinity chromatography on immobilized Cratylia mollis isolectins

A highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E. paniculatus seed flour (25 gl(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28 degrees C) was chromatographed on Sephadex G-25 (12 mlh(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). The column chromatography was performed at 24 degrees C; the matrix was washed (30 mlh(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography.     

Purification and Characterization of the polygalacturo nase from Tomato Fruits

The polygalacturonase (PG) isolated fron the pericarp of fully ripe tomato fruits was purified by (NH4)2SO4 precipitation, carboxyme-thylsepharose ion-exchang column and sephadex G-75 gel filtration. Specific activity of purified PG was 1.5 μmol galacturonic acid mg-1 protein min-1, which was 30 times as high as that of the crude extract with 1.7mol NaC1. When the elution separated by second sephadex G-75 column was analyzed by SDS-PAGE, only a single protein band was detected. It was shown by heat and pH experiments of the purified enzyme that the enzyme activity retained 50%, after treatment with heat at 50℃ for 10 min, and that the optimal pH was 4.6.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.