Yeast Glycogen Phosphorylase: Characterization of the Dimeric Form and Its Activation

The purification of yeast glycogen phosphorylase [EC 2.4.1.1] was improved by ethanol precipitation and affinity chromatography on a glycogen-Sepharose column. The purified enzyme gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a subunit molecular mass of 100 kDa. Gel electrophoresis also showed that the major activity of native phosphorylase was ascribed to a dimer of 203 kDa, which was agreed with the value obtained by gel filtration on Sephadex G-200. The yeast phosphorylase showed a high affinity for AMP- Sepharose, whereas the enzyme was specifically inhibited by AMP. This inhibition was competitive with respect to the substrate glucose 1-phosphate and gave a Ki value of 9.3 mm. Activation of the crude extract by phosphorylation with an endogenous phosphorylase kinase indicated that the yeast phosphorylase occurred in a mixture of phosphorylated and non-phosphorylated forms.     

Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits.

Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) from fresh human erythrocytes has been purified to homogeneity in two steps with an overall yield of 56%. The purification involves DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharose/formycin B. This scheme is suitable for purification of the phosphorylase from as little as 0.1 ml of packed erythrocytes. The native enzyme appears to be a trimer with native molecular weight of 93,800 and the subunit molecular weight of 29,700 +/- 1,100. Two-dimensional gel electrophoresis of the purified enzyme under denaturing conditions revealed four major separable subunits (numbered 1 to 4) with the same molecular weight. The apparent isoelectric points of subunits 1 to 4 in 9.5 M urea are 6.63, 6.41, 6.29, and 6.20, respectively. The different subunits are likely the result of post-translational modification of the enzyme and provide an explanation of the complex native isoelectric focusing pattern of purine nucleoside phosphorylase from erythrocytes. Three of the four subunits are detectable in two-dimensional electrophoretic gels of crude hemolysates. Knowing the location of the subunits of purine nucleoside phosphorylase in a two-dimensional electropherogram allows one to characterize the purine nucleoside phosphorylase in crude cell extracts from individuals with variant or mutant purine nucleoside phosphorylase as demonstrated in a subsequent communication. Partial purification of the phosphorylase from 1 ml of erythrocytes on DEAE-Sephadex increases the sensitivity of detection of the subunits to the 0.3% level.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.

A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.     

Isolation and characteristics of functionally active proteolytically modified forms of tyrosyl-tRNA synthetase from bovine liver

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).     

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.     

1,4-alpha-Glucan phosphorylase from the slime mold Physarum polycephalum. Purification, physico-chemical and kinetic properties

Glycogen phosphorylase from macroplasmodia of Physarum polycephalum was purified 76-fold to homogeneity. The native enzyme migrated as a single protein band on analytical disc gel electrophoresis coinciding with phosphorylase activity. After reduction in the presence of sodium dodecylsulfate one protein band was detectable which corresponded to an Mr of 93 000. The molecular weight of the native enzyme determined by gel sieving or gradient-polyacrylamide gel electrophoresis was 172000 and 186000, respectively. The enzyme contained about 1 mol pyridoxal 5'-phosphate and less than 0.1 mol covalently bound phosphate per mol subunit. The amino acid composition of the enzyme was determined. In the direction of phosphorolysis the kinetic data were determined by initial velocity studies, assuming a rapid equilibrium random mechanism. Glucose 1-phosphate and GDP-glucose were competitive inhibitors toward phosphate and noncompetitive to glycogen. 5'-AMP, a weak activator of the enzyme, counteracted the glucose-1-phosphate inhibition completely. Physarum phosphorylase was compared with phosphorylases from other sources on the basis of chemical and kinetic properties. No evidence for the presence of phosphorylated forms has yet been found.     

Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves

The non-chloroplastic -glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetic acid - G1P glucose 1-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - PMSF phenylmethyl sulphonyl fluoride - SDS sodium dodecylsulfate - Tris Tris (hydroxymethyl)aminomethane Dedicated to Professor Dr. A. Pirson on the occasion of his 70th birthday     

Purine nucleoside phosphorylase of the malarial parasite, Plasmodium lophurae

Purine nucleoside phosphorylase (EC 2.4.2.1, purine nucleoside:orthophosphate ribosyltransferase) was purified and characterized from the malarial parasite, Plasmodium lophurae, using a chromatofocusing (Pharmacia) column and a formycin B affinity column. The apparent isoelectric point of the native protein, as determined by chromatofocusing, was 6.80. By gel filtration and both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the native enzyme appeared to be a pentamer with a native molecular weight of 125,300 and a subunit molecular weight of 23,900. The enzyme had a broad pH optimum, pH 5.5-7.5, with maximum activity at pH 6.0-6.5. The enzyme reaction was readily reversible with a Km for inosine of 33 microM and a Km for hypoxanthine of 82 microM. Thioinosine, guanosine, and guanine were also substrates for the plasmodial enzyme, but allopurinol and adenine were not. The parasite enzyme was competitively inhibited by formycin B (Ki = 0.39 microM). Formycin A, azaguanine, and 8-aminoguanosine were not inhibitors of the enzyme.     

Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV

Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.     

1, 4-alpha-Glucan phosphorylase from Klebsiella pneumoniae purification, subunit structure and amino acid composition.

1. A 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae has been purified about 80-fold with an over-all yield greater than 35%. The purified enzyme has been shown to be homogeneous by gel electrophoresis at different pH-values, by isoelectric focusing, by dodecylsulfate electrophoresis and by ultracentrifugation. 2. The molecular weight of the native enzyme has been determined to be 180 000 by ultra-centrifugation studies, in good agreement with the value of 189 000 estimated by gel permeation chromatography. 3. The enzyme dissociates in the presence of 0.1% dodecylsulfate or 5 M guanidine hydrochloride into polypeptide chains. The molecular weight of these polypeptide chains has been found to be 88 000 by dodecylsulfate polyacrylamide gel electrophoresis and 99 000 by sedimentation equilibrium studies, indicating that the native enzyme is composed of two polypeptide chains. 4. The enzyme contains pyridoxalphosphate with a stoichiometry of two moles per 180 000 g protein, confirming that the 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is a dimeric enzyme. 5. The amino acid composition of the enzyme has been determined, and its correspondence to that of 1,4-alpha-glucan phosphorylases from other sources is discussed. 6. The pI of the enzyme has been shown to be 5.3 and its pH-optimum to be about pH 5.9. The enzyme is stable in the range from pH 5.9 to 10.5.     

Beta-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme

A beta-D-glycosidase activity was purified from mycelium of Humicola grisea var. thermoidea grown on avicel as the main carbon source. The purified enzyme was a glycoprotein and migrated as a single polypeptide band on polyacrylamide gel electrophoresis under native or denaturing conditions. The apparent molecular weight of the enzyme was estimated to be 55 kDa by gel filtration and SDS-PAGE. The enzyme was active against o-nitrophenyl beta-D-galactoside; p-nitrophenyl beta-D-glucoside, p-nitrophenyl beta-D-fucoside, lactose and cellobiose, PNP fucoside (synthetic substrate) and cellobiose (natural substrate) being the best utilized. A comparison of the properties of beta-D-galactosidase, beta-D-glucosidase and beta-D-fucosidase showed that three activities exhibited similar pH and temperature optima and the same thermostability. The hydrolysis rate of substrate mixtures suggests that the enzyme possesses a common catalytic site for all the substrates assayed.     

Purification and properties of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

Blue crab muscle (Callinectes danae) glycogen phosphorylase a was purified by adsorption of a crude extract on a starch column, elution with a dilute glycogen solution, selective precipitation with ammonium sulfate, dialysis against a solution containing ammonium sulfate and ethylenediaminetetraacetate, followed by centrifugation and chromatography on Sephadex G-25 (sp act 64.5 IU, recovery of 53.8%, and a purification factor of 189). The lyophilized preparation is stable for several months. Disc electrophoresis of the purified phosphorylase yields two protein bands, both with enzymatic activity of the a form. One of the protein bands represents about 10% of the total amount of protein present in the two bands. The molecular weight of the enzyme is 176,000 as determined by ultracentrifugation in a sucrose density gradient and 180,000 as determined by discontinuous polyacrylamide gel electrophoresis. The molecular weight found by disc electrophoresis corresponds to the main protein band. Crab muscle phosphorylase a is not associated under electrophoretic conditions in which rabbit muscle phosphorylase a shows association behavior. Subunit studies by continuous SDS-gel electrophoresis suggest that crab muscle phosphorylase a possesses only one subunit. Pyridoxal-5′-phosphate is a cofactor of the enzyme.     

Detection of Glycogen-Debranching System in Trophozoites of Entamoeba histolytica

ABSTRACT. Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4- α -glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl- α -glucoside yielding successively 4-nitrophenyl- α -maltoside and 4-nitrophenyl- α -maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of M_r= 180,000 ± 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Light-mediated changes in the plastidic phosphorylase patterns in shoots of Pisum sativum

α-1,4-Glucan phosphorylase (EC 2.4.1.1) forms from light or dark grown shoots of Pisum sativum L. cv. 'Kleine Rheinländerin' have been studied using various electrophoretic techniques. The phosphorylase patterns of green and etiolated shoots differed. Etiolated shoots contained two enzyme forms, one residing inside and the other outside the etioplast; this was shown by electrophoresis of extracts of isolated etioplasts. Purity and intactness of the organelle preparation were ascertained by electron microscopy. Light-grown shoots contained, in addition to these two enzyme forms, a third phosphorylase which appears to be chloroplast-specific. The two plastidic phosphorylase forms differed slightly in their apparent molecular masses (as determined by non-denaturing polyacrylamide gel electrophoresis) and in their affinities towards branched polyglucans (as revealed by affinity electrophoresis). The apparent affinity of the extrachloroplastic phosphorylase form to these polyglucans was orders of magnitude higher than that of the two plastidic enzyme forms. The development of the chloroplast-specific phosphorylase pattern is under photocontrol. Investigations performed with red or far-red illuminated wild-type plants and with a pale mutant which has a highly reduced pigment and thylakoid content suggest that this photocontrol is mediated by phytochrome.     

Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization,developmental change,in-vitro translation,and processing

The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of Pisum sativum L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa).Abbreviations IgG immunoglobulin G - kDa kilodalton - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehüser for help in the immunocytochemical studies.     

Purification and partial characterization of recombinant human differentiation-stimulating factor

Recombinant human differentiation-stimulating factor (rhD-factor) has been isolated to greater than 95% purity from Chinese hamster ovary cells. RhD-factor is a glycoprotein with an apparent molecular weight of 45.6 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration in 6 M guanidine-hydrochloride, rhD-factor elutes with an apparent molecular weight of 21.5 kDa; it elutes with an apparent molecular weight of 44.8 kDa under neutral pH (native) conditions. The amino-terminal sequence (12 residues) is consistent with the expected sequence derived from the genomic DNA sequence. Recombinant D-factor is heavily glycosylated with 30% by weight neutral sugar and 12% sialic acid. The ED50 for rhD-factor was 0.25 ng/ml. Trifluoromethanesulfonic acid-deglycosylated rhD-factor has a biological activity comparable to that of the native recombinant protein (ED50 = 0.40 ng/ml). The biological activity of rhD-factor was stable at pH 1 for 40 h, in 6 M guanidine-HCl containing buffers with or without reducing agent, and in 1% SDS. Carboxymethylation of D-factor after reduction totally destroyed biological activity.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Purification and characterization of Aspergillus ficuum endoinulinase

Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.