High resolution of peroxidase-indoleacetic Acid oxidase isoenzymes from horseradish by isoelectric focusing

Several improved techniques for isoelectric focusing of isoenzymes in polyacrylamide gel slabs were developed. Using these techniques, three commercial sources of horseradish peroxidase were each examined with three commercial sources of carrier ampholytes to determine their respective isoenzyme profiles.     

The separation of adenine and hypoxanthine-guanine phosphoribosyl transferases isoenzymes by disc gel electrophoresis

Hypoxanthine-guanine (HGPRT; E.C. 2.4.2.8) and adenine (APRT; E.C. 2.4.2.7) phosphoribosyl transferases were studied by disc electrophoresis on polyacrylamide gel. The positions of the isoenzymes were detected by radiochemical enzyme assay. The nucleotide products of the reactions were precipitated in the gel with lanthanum chloride. APRT was found to migrate slightly less rapidly than albumin and produced a single narrow symmetrical peak of activity. HGPRT migrated 25–50% more slowly than albumin and produced a broad zone of activity consisting of four unequal peaks. The APRT enzyme of Rhesus monkey liver and the HGPRT enzyme of sheep erythrocytes migrated notably slower than the corresponding human enzymes. An isoenzyme of APRT was detected in human erythrocytes which migrated more rapidly than that of most individuals. In all instances, the adenine was utilized by one electrophoretic component and hypoxanthine and guanine by another. Furthermore, the components which utilized hypoxanthine and guanine were inseparable. The sensitivity of the assay made it possible to assess the electrophoretic and enzymatic characteristics of HGPRT isoenzymes on aliquots of hemolysates capable of producing 0.5 picomoles of IMP per minute. In human erythrocytes with normal enzyme content, this amount of activity is present in approximately 50 nanoliters of cells.Aided by U.S. Public Health Service grants Nos. HD 04608 and HD 03015 from the National Institute of Child Health and Human Development, National Institutes of Health.     

Scanning Isoelectric Focusing and Isotachophoresis of Clostridium perfringens Type A Enterotoxin

Fractionation of highly purified Cl. perfringens type A enterotoxin by scanning isoelectric focusing (SIF) and isotachophoresis (IT) in polyacrylamide gels is described for the first time. The use of 2% ampholytes pH 3–6 allowed the separation of enterotoxin into 2 species. The major component had an isoelectric point of 4·5 and possessed antigenic as well as functional activity. The minor component of enterotoxin, at equivalent concentrations, was devoid of any demonstrable biological activity had an isoelectric point of 4·6 and appeared to represent approximately 15% of the purified enterotoxin. With ampholytes pH 3·5–10 the minor and major components were focused at different times than when ampholine pH 3–6 was employed. Electrofocusing of enterotoxin in the presence of 6 M-urea did not alter the SIF pattern. During IT the major component of enterotoxin migrated ahead of the minor component. The 2 proteins were completely separated. Isotachophoretic separations required 0·023 M-phosphate pH 6·0 as the leading ion, 0·079 M-Tris as the counter-ion, 0·2 M-glycine (in Tris pH 8·1) as the terminating ion, 30 γ carrier ampholytes pH 3·5–10, 263 μg enterotoxin, 4% acrylamide and a current of 5 mA per gel column.     

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.     

Human brain hypoxanthine guanine phosphoribosyltransferase: structural and functional comparison with erythrocyte hypoxanthine guanine phosphoribosyltransferase

A rapid and simple method, based on GMP Sepharose affinity chromatography, was used for the purification of human brain hypoxanthine guanine phosphoribosyltransferase. A single protein band was detected by polyacrylamide gel electrophoresis of the native purified enzyme. A subunit molecular weight of 25,000 was estimated by SDS gel electrophoresis. The Km values for hypoxanthine and phosphoribosyl pyrophosphate were 50 and 111 microM, respectively. The Ki values for GMP and IMP with phosphoribosyl pyrophosphate were 21 and 37 microM, respectively. The purified enzyme from human brain did not differ significantly from the human erythrocyte one in amino acid composition. The brain and erythrocyte hypoxanthine guanine phosphoribosyltransferases showed complete immunochemical identity on Ouchterlony double diffusion.     

Purification and characterization of a lysosomal form and a variant form of beta-glucuronidase from the rat basophil leukaemia tumour.

Two isoenzyme of beta-glucuronidase from a rat basophil leukaemia tumour were co-purified 4067-fold by (NH4)2SO4 precipitation and sequential chromatography on concanavalin A--Sepharose, Sephadex G-200, DEAE-cellulose, CM-cellulose and phosphocellulose. The purity of the mixture was established by the coincidence of the peaks of enzyme activity and protein at a molecular weight of 300 000 on Bio-Gel P-300, the presence of only two protein bands, both of them enzymically active, in polyacrylamide gels after electrophoresis under non-denaturing conditions, and the presence of a single subunit species, of mol.wt. 75 000, after electrophoresis in polyacrylamide gels under a denaturing conditioning. The major isoenzyme co-migrated with the L form from rat liver during electrophoresis in alkaline polyacrylamide gels, whereas the minor isoenzyme migrated more rapidly than either the lysosomal form or the rat liver microsomal form and was designated the tumour (T) isoenzyme. A mixture of the purified isoenzymes from two preparations had an average specific activity of 1389 units/mg for phenolphthalein beta-D-glycopyranosiduronic acid. The L and T isoenzymes, which had pI5.9 and 5.7 respectively, could be obtained free of cross-contamination by isoelectric focusing and had similar specific activities. Although the T isoenzyme could be a catabolic product of the M or the L form, it could also be a unique tumour product, because it was not detected in extracts of normal rat tissues.     

Isolation and properties of multiple forms of histidine decarboxylase from rat gastric mucosa.

The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.     

Glutathione transferase from bovine placenta. Preparation, biochemical characterization, crystallization, and preliminary crystallographic analysis of a neutral class PI enzyme

A method was developed to purify glutathione transferase from bovine placenta by affinity chromatography and fast protein liquid chromatography. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. The dimeric enzyme is composed of identical subunits with a molecular weight of 23,000; its isoelectric point is 6.9. In contrast to previously described isoenzymes of glutathione transferase, the protein we have purified exists in two forms, an active reduced form and a less active oxidized form. These can be reversibly transformed into each other but behave differently in sedimentation analysis, gel chromatography, and gel electrophoresis. These differences may reflect a change in the molecular shape of glutathione transferase. Chemical modification with iodoacetate, iodoacetamide (presumably of thiol groups), phenylglyoxal, and butadione (presumably of arginyl groups), and their inhibitory effects on the activity were investigated. From substrate specificity studies and N-terminal sequence analysis it is obvious that this glutathione transferase must belong to the isoenzyme class pi. The purified enzyme could be crystallized from 1.4 M ammonium sulfate solution, pH 8.0, in the presence of S-hexyl-glutathione. The crystals are tetragonal, with space group P4(1)2(1)2 or P4(3)2(1)2. The cell constants are a = b = 6.1 nm, c = 23.7 nm, alpha = beta = gamma = 90 degrees. The crystals diffract to 0.26-nm resolution and are suitable for x-ray structure analysis.     

Plant microbody proteins, I. Purification and characterization of catalase from leaves of lens culinaris.

1) Catalase from green leaves of Lens culinaris (lentils) was investigated with respect to isoenzyme patterns. In contrast to other plants, which have been reported to contain multiple forms of catalase, only one form of this enzyme was revealed when crude extracts were subjected to starch gel electrophoresis or to polyacrylamide disc-gel electrophoresis. Furthermore, catalases from leaves, stems and cotyledons were electrophoretically identical. 2) The leaf enzyme has been purified by conventional methods to apparent homogeneity. It has a molecular weight of 225 000 (ultracentrifuge) and is composed of four identical subunits of molecular weight 54 000 (sodium dodecylsulphate gel electrophoresis). The ratio A280/A405 of the pure enzyme was found to be 1.5. The isoelectric point is at pH 5.5. The enzyme, very labile at pH-values below 7.0, is stable in Tris chloride and potassium phosphate buffers between pH 7.5 and 9.5. It is slowly inactivated by 1mM dithiothreitol and is rapidly inactivated by 1mM mercaptoethanol. 3) The catalase was shown to be the major protein component of the peroxisomal matrix. It could not be detected at the membranes of the leaf peroxisomes.     

Separation, purification, and comparative properties of chloroplast and cytoplasmic phosphoglycerate kinase from barley leaves

The chloroplast and cytoplasmic isoenzymes of phosphoglycerate kinase (PGK) (EC. 2.7.2.3) from Hordeum vulgare leaves have been separated and purified for the first time to apparent homogeneity. The method for purifying the isoenzymes is described here and consists of DEAE Sephacel chromatography followed by affinity chromatography on ATP Sepharose. This consistently provided a 500- to 900-fold purification of each isoenzyme. Most of the total PGK in green barley leaves was found to be in the chloroplasts with only 10% in the cytoplasm. The immunological properties of the two isoenzymes were compared. The antisera raised to the separate isoenzymes showed cross-reactivity, although there is evidence that each isoenzyme possesses some distinct epitopes. The isoenzymes differ in overall charge with isoelectric points at 5.2 and 5.4 for the chloroplast and cytoplasmic isoenzymes, respectively. Molecular mass estimations by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis provided similar values of approximately 38 kilodaltons for each isoenzyme, some 4 to 5 kilodaltons less than the values calculated from the cDNA sequences of the wheat isoenzymes. The isoenzymes have broadly similar pH optima of pH 7 to 8. The cytoplasmic isoenzyme is more thermally stable than the chloroplast isoenzyme. Further studies are now in progress to compare both the regulatory properties of the isoenzymes and also their three-dimensional structures as compared with the yeast enzyme.     

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.     

A rapid purification procedure for camel kidney glutathione S-transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37,000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 mumol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100. The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29,000 D and 26,000 D to give a native molecular weight of 55,000 D. The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. 1-chloro-2,4-dinitrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse. Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Isolation and characterization of glycogen branching enzyme from rabbit liver

Glycogen branching enzyme was isolated from rabbit liver. The highly purified enzyme shows a monomer molecular weight of 71 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and apparent molecular weights of 93 000 by sucrose density gradient sedimentation and 52 000 by gel-exclusion chromatography on Sephacryl S-300. No glucosamine, mannosamine, galactosamine, or sialic acid was detected in the protein. An amino acid analysis is reported. The spectrum of branching enzyme is that of a simple polypeptide, with A1%280nm = 24.6. Highly purified branching enzyme consists of several closely related active enzyme forms that can be resolved by isoelectric focusing in polyacrylamide gel. The major species of pI 5.7 is flanked by less abundant forms of pI 5.6 and 5.8. Seemingly identical enzyme forms are observed in crude extracts of rabbit liver, skeletal muscle, brain, and heart, although the absolute and relative concentrations vary among the tissues. Branching enzyme apparently does not exhibit tissue-specific isoenzymes.     

Purified guanylate cyclase: characterization, iodination and preparation of monoclonal antibodies

Guanylate cyclase was purified from the soluble fraction of rat lung using a modification of procedures published previously. The purified enzyme exhibited specific activities, at pH 7.6, of 219-438 nmoles/mg protein/min and 34-60 nmoles/mg protein/min with Mn2+ and Mg2+ as cation cofactors, respectively. The specific activity changed as a function of the protein concentration due to a change in Vmax with no alteration of the Km for GTP. The enzyme migrated as a single band coincident wih guanylate cyclase activity on nondenaturing polyacrylamide and isoelectric focusing gels (isoelectric point = 5.9). Purified guanylate cyclase had an apparent molecular weight of 150,000 daltons as determined by gel filtration chromatography and polyacrylamide gel electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate revealed a single subunit of 72,000 daltons, suggesting that the enzyme is a dimer of an identical subunit. The purified enzyme could be activated by nitric oxide, indicating that this compound interacts directly with the enzyme.     

Cathepsin D. Purification of isoenzymes from human and chicken liver

1. The Barrett (1967) assay for cathepsin D was slightly modified. 2. The enzyme was purified from liver of man and chicken by a procedure involving autolysis, acetone fractionation, ion-exchange chromatography and isoelectric focusing. 3. Several isoenzymes of cathepsin D were resolved in the isoelectric-focusing step, and three major forms, alpha,beta and gamma, were distinguished for each species. 4. A modified analytical method of isoelectric focusing in polyacrylamide gel indicated a high degree of homogeneity of the purified beta and gamma isoenzymes from each species, and this was supported by their constant high specific activities. 5. Gel filtration of the isoenzymes in a calibrated column of Sephadex G-100 showed that each had a molecular weight of 45000. 6. Human cathepsin D had a pH optimum of 3.5, and that of chicken enzyme was 3.0, haemoglobin being used as substrate. In each species, the three isoenzymes have the same pH-dependence curve. 7. The purified cathepsin D samples showed very little action on acid-denatured albumin.     

Isolation of Active cAMP Dependent Protein Kinases from Calf Ovaries: Gel Electrcphoresis vs. Gel Electrofocusting

Calf ovarian CAMP dependent protein kinase A was isolanted by adsorntion on to DEAF-cellulose, gel chromatography on agarosepolyacrylamide copolymer, electrophoresis in a 6% polyacrylamide gel, 0.2% in Iriton X-100, and DEAF-chromatography. The yield was 3.3 mg, representinw 22% of the starting material.

Purification was 400-fold. The product appears homogeneous on gel electropheresis at p 10.2, but DEAF-chromatography, gel elecectro focusing and gel electrophoresis at pH 8.5 and 7.5 reveal two charge isomeric forms of the enzyme.

Optimization of gel concentration for the separation of the enzyme from its closest migrating contaminant pointed to gel electrofocusing, rather than electrophoresis, as the appropriate Separation tool. However, that electrophoresis, as the inactivate the enzyme when conducted on wide-diameter preparative gels, if allowed to proceed to the steady-state, using either Ampholine or buffers as the carrier ampholytes, and etyleneglycol to repress no I yacrylami decopolyner, electrophorus is in a 6 po lyacrylaniide el, 0.7 in TM on -1 00, and DSAF-chromatography. The yield was 3.1 me, representing 787% of the starting material Petrification was 400-fold. The product appears homos jeneousonge I electrophoresis at pM 10.7, hut DEAF- chromatography, gel electron focus inn and ruels ectroohores is at pH 6.5 and 7.5 reveal two charred is on ericforirs of the enzyme, Opticalization of feel concentration for the separation of the enzyme from its closest ml floating cont eminent pointed to jel electro focusing, rather then elect rophoresis, as the appropriate separation tool. However, that method proved to inactivate the enzyme when c on ducted on wide-diameter preparative gels, if all owed to proceed to the steady-state, usinp; either Ampholine or buffers as the carrier ampholytes, and ethyl eneglycol to repress isoelectric precipitation. Only buffer electrofoucing on ultrogel Aca 54 if stoppert prior to the attainment of the isoelectric endpoint of the enzyme succeeded in recovering substantial (65%) activity, albeit at the price of resolution. Thus, a non-optimal concentration in polyacrylamide gel electrophoresis was applied in preference to preparative gel electro focusing.

Preparative methods for the isolation of Proter Kinase 8 and CAMP Binding Pritein A in homogeneous form were also developed, using nodifications of the above-stated procedure.     

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.