Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis.

A methyltransferase that methylates one of the proteins involved in chemotactic adaptation to sensory stimuli in Bacillus subtilis was purified to homogeneity. The enzyme utilizes S-adenosylmethionine as donor for a methyl group that is transferred to a glutamate residue in a 69 000-mol.wt. membrane protein and also to a protein of 19 000 mol.wt. The molecular weights of the denatured enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of the native enzyme by gel-filtration chromatography both show the protein to be a 44 000-mol.wt. monomer. Isoelectric focusing of the purified methyltransferase showed the protein to be a single species with isoelectric point pI 5.4. On the basis of a molecular weight of 44 000, the molar absorption coefficient at 262 nm of the enzyme is 10.9 x 10(4) M-1 . cm-1. The Km of the enzyme for S-adenosylmethionine is about 2 microM. The Ki for S-adenosylhomocysteine is about 0.2 microM. Ca2+ is a competitive inhibitor of methylation, with a Ki of 0.065 microM. The enzyme methylates membranes from the wild-type more efficiently than membranes isolated from a mutant strain defective in chemotaxis. The enzyme is unable to methylate Escherichia coli membranes.     

Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the branching enzyme

A branching enzyme was extracted from the mycelia of Neurospora crassa and was purified to electrophoretic homogeneity by procedures including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose 4B column chromatography and gel filtration on Toyopearl HW-55S. The final yield of the branching enzyme activity was 15.1%, and the final purified enzyme preparation showed a specific activity of 702 units per mg of protein. The molecular weight of this enzyme was estimated to be 80,000 by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. The amino acid composition and the carbohydrate content of this enzyme were analyzed. The isoelectric point of this enzyme determined by polyacrylamide gel isoelectrofocusing was 5.6. The branching activity of the enzyme was confirmed by its action on amylopectin as well as by the combined action of this enzyme and N. crassa glycogen synthase. The action of this enzyme on amylopectin decreased the wavelength of the absorption maximum of the glucan-iodine complex, and increased the amount of the short unit chains of the debranched product. The product obtained by the combined action yielded beta-limit dextrin upon hydrolysis with beta-amylase. No multiplicity was found for the branching activity either by chromatography or by electrophoresis.     

Purification of S-adenosylmethionine decarboxylase from soybean.

S-Adenosylmethionine decarboxylase (EC 4.1.1.19) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone)-Sepharose 6B chromatographies. The enzyme was free from diamine oxidase activity. The molecular weight of the enzyme estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was 66,000. The Km value for S-adenosylmethionine was 0.26 mM. The optimum pH and temperature were 7.5 and 40 degrees C. Neither putrescine nor Mg2+ affected the enzyme activity, but the enzyme was inhibited by spermidine, spermine, methylglyoxalbis(guanylhydrazone), sodium borohydride and phenylhydrazine. Agmatine was a novel inhibitor which inhibited S-adenosylmethionine decarboxylase and arginine decarboxylase, preventing the accumulation of decarboxylated S-adenosylmethionine and putrescine, respectively.     

Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.     

A new purification procedure for bovine milk xanthine oxidase: effect of proteolysis on the subunit structure.

A new purification procedure for bovine milk xanthine oxidase is reported. The enzyme so obtained is of the highest purity and shows little evidence of degradation. The enzyme displays a single protein band on either polyacrylamide gels or on sodium dodecyl sulfate-urea polyacrylamide gels. Sedimentation equilibrium studies indicate a native molecular weight of 303,000 and a subunit molecular weight of approximately 150,000. The latter value is in good agreement with the minimum molecular weight of 157,000 calculated from dry weight determination and flavin analysis. In contrast, purification of xanthine oxidase from pancreatin-treated cream yields a protein which displays two subunits corresponding to molecular weights of 92,000 and 39,000 as determined by dodecyl sulfate-urea polyacrylamide gel electrophoresis. Pancreatinized enzyme has a greater mobility than unproteolyzed enzyme on polyacrylamide gels. Exposure of milk xanthine oxidase to pancreatin before isolation or after purification yields the same result.     

Purification and characterization of a heme-containing amine dehydrogenase from Pseudomonas putida.

The primary amine dehydrogenase of Pseudomonas putida NP was purified to homogeneity as judged by polyacrylamide gel electrophoresis. Cytochrome c or an artificial electron acceptor was required for amine dehydrogenase activity. The enzyme was nonspecific, readily oxidizing primary monoamines, benzylamine, and tyramine; little or no measurable activity was detected with isoamines, L-ornithine, L-lysine, and certain diamines or polyamines. The pH optima for n-butylamine, benzylamine, and n-propylamine were 7.0, 6.5, and 7.0, respectively. The molecular weight of the enzyme was 112,000 as determined by gel filtration and 95,300 as analyzed by sedimentation equilibrium. Subunit analysis by sodium dodecyl sulfate gel electrophoresis suggested that the enzyme was composed of two nonidentical subunits with molecular weights of 58,000 and 42,000. The absorption spectrum of the purified enzyme was indicative of a hemoprotein, exhibiting absorption maxima at 277, 355, and 408 nm. Reduction with sodium dithionite or amine substrates resulted in absorption maxima at 523 and 552 nm and a shift in the Soret peak to 416 nm. These results suggested that the enzyme is a hemoprotein of the type c cytochrome. There was no evidence that flavins were present.     

S-adenosyl-L-methionine:thioether S-methyltransferase, a new enzyme in sulfur and selenium metabolism

The final urinary excretion product of selenium detoxification is trimethylselenonium ion. An assay has been developed for the enzyme, S-adenosylmethionine:thioether S-methyltransferase, responsible for this final methylation reaction. This assay employed high pressure liquid chromatography separation and quantitation of the trimethylselenonium ion produced by thioether methyltransferase acting on S-adenosylmethionine and dimethyl selenide. The enzyme was shown to reside primarily in the cytosol of mouse lung (30 pmol/mg protein/min) and liver (7 pmol/mg protein/min). Purification from mouse lung to a preparation that exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was achieved by DEAE, gel filtration, and chromatofocusing chromatographies. Thioether methyltransferase is monomeric with a molecular weight of 28,000 and has a pI of 5.3. The pH optimum was 6.3, and Km values for dimethyl selenide and S-adenosylmethionine were 0.4 and 1.0 microM, respectively. The enzyme was inhibited 50% by 25 microM sinefungin, an analog of S-adenosylmethionine, or 40 microM S-adenosylhomocysteine, the reaction product. Pure thioether methyltransferase methylated selenium in dimethyl selenide, tellurium in dimethyl telluride, and S in dimethyl sulfide and many other thioethers. These data suggest a general role for this novel enzyme in the synthesis of onium compounds with increased aqueous solubility helpful in their excretion.     

Purification and molecular identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme

Two different molecular species of protein methylases I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23), one specific for myelin basic protein (MBP) and the other for histone, have been purified from calf brain to near homogeneity, as discerned by nondenaturing polyacrylamide gel electrophoresis. Although both methylases share some common properties, such as utilization of S-adenosyl-L-methionine as the methyl donor and methylation of protein-bound arginine residues, they are distinctly different from each other in molecular weight and in catalytic, as well as the immunological, properties. The MBP-specific protein methylase I (approximately 500 kDa) methylates MBP preferentially (Km = 2 X 10(-7) M) and histone to a much lesser extent (Km = 1 X 10(-4) M), while the histone-specific methylase I (approximately 275 kDa) methylates histone only. Both methylases exhibit two major subunit bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 100 and 72 kDa for the MBP-specific and 110 and 75 kDa for the histone-specific. At 0.5 mM p-chloromercuribenzoate, about 50% of the MBP-specific enzyme remained as active, while most of the histone-specific enzyme activity was lost. In 2 mM guanidine HCl, approximately 90% of the former enzyme activity remained while nearly complete inactivation of the latter enzyme was observed. The enzymes also exhibited quite different inactivation profiles toward high temperature (45-65 degrees C); MBP-enzyme was stable up to 50 degrees C and was rapidly inactivated at higher temperatures with an inflection point at about 57 degrees C. However, under the identical conditions, histone-enzyme was inactivated progressively and linearly in the same temperature range. Finally, Western immunoblot analysis of polyclonal antibodies directed against either enzyme exhibited no cross-reactivity with the other.     

Preparation and some properties of homogeneous Neurospora crassa assimilatory NADPH-nitrite reductase.

The Neurospora crassa assimilatory NADPH-nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4), which catalyzes the NADPH-dependent formation of ammonia from nitrite, has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme is 26.9 mumol nitrite reduced/min per mg protein, which corresponds to a turnover number of 7800 min(-1). The enzyme also has associated NADH-nitrite reductase, NADPH-hydroxylamine reductase and NADH-hydroxylamine reductase activities. The stoichiometry of 3 mol NADPH oxidized per mol nitrite reduced and ammonia formed has been confirmed. The visible absorption spectrum of the nitrite reductase reveals maxima at 280,390 (Soret) and 580 (alpha) nm. The latter bands are indicative of the occurrence of siroheme as a prosthetic group. The A280nm/A390nm ratio of 7.0 and the Soret/alpha ratio of 3.8 are compatible with values reported for other purified siroheme-containing enzymes. These results are discussed in terms of the comparative biochemistry of various enzymes involved in nitrite, hydroxylamine and sulfite metabolism in Neurospora crassa and other organisms.     

Purification of phosphatidylethanolamine N-methyltransferase from rat liver

Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine.     

Protein and lipid methylation by methionine and S-adenosylmethionine in Myxococcus xanthus

Methylation of lipids and proteins has been examined in Myxococcus xanthus using radioactive methionine and S-adenosylmethionine as methyl donors. S-adenosylmethionine is shown to be taken up by these cells and utilized directly. This permits detection of methylation in the presence of protein synthesis. Patterns of methylation obtained using methionine and S-adenosylmethionine during vegetative growth are compared by polyacrylamide gel electrophoresis, and inhibitors of protein synthesis and S-adenosylmethionine synthesis are examined for their effects on methylation. The ability to investigate methylation using exogenous S-adenosylmethionine will be advantageous in studying the role of methylation under conditions of growth and development where ongoing protein synthesis is required.     

Purification and properties of 2-aminoethylphosphonate:pyruvate aminotransferase from Pseudomonas aeruginosa

2-Aminoethylphosphonate aminotransferase has been purified to homogeneity with a yield of 15% from cell extracts of Pseudomonas aeruginosa. The molecular weight of the enzyme was estimated by gel filtration to be 65000 +/- 2000. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a molecular weight of 16500 +/- 1000, suggesting a tetrameric model for this protein. The absorption spectrum exhibits maxima at 280 nm, 335 nm and 415 nm which are characteristic of a pyridoxal-phosphate-dependent enzyme: 4 mol of pyridoxal 5'-phosphate/mol of enzyme have been found. This aminotransferase catalyzes the transfer of the amino group of 2-aminoethylphosphonate (ciliatine) to pyruvate to give 2-phosphonoacetaldehyde and alanine. A pH optimum between 8.5-9 and an activity increasing from 30 degrees C to 50 degrees C have been observed. The reaction follows Michaelis-Menten kinetics with Km values of 3.85 mM and 3.5 mM for ciliatine and pyruvate respectively. This enzyme shows a very high specificity since ciliatine and pyruvate are the only amino donor and acceptor respectively. Methyl, ethyl and propylphosphonic acids are better competitors towards ciliatine than their alpha-amino derivatives. 3-Aminopropylphosphonate, the higher homologue of ciliatine, is recognized neither as a substrate nor as an inhibitor. The enzyme activity is significantly affected by carbonyl reagents and by HgCl2. Transamination of 2-aminoethylphosphonate is the first step of a double-step pathway which leads to the cleavage of its C-P bond.     

Diamine oxidase: molecular weight and subunit analysis.

Electrophoretically pure hog kidney diamine oxidase has been isolated by an improved procedure and subjected to molecular weight and subunit analyses. Sedimentation/diffusion and sedimentation equilibrium ultracentrifugation clearly show that the native enzyme has a molecular weight of 172,000. Acrylamide gel electrophoresis indicates that the enzyme consists of two apparently identical subunits of 91,000 daltons each. The native enzyme contains two firmly bound Cu(II) ions. The isolation procedure described provides diamine oxidase in 50–60% yield of activity and of the highest specific activity yet reported (1.2 units/mg).     

Purification and characterization of poly(ADP-ribose) synthetase from calf thymus.

Poly(ADP-ribose) synthetase from calf thymus has been purified to apparent homogeneity by a simple and rapid method with a recovery of 10 to 20%. The enzyme activity absolutely requires the presence of DNA. Histone further stimulates the reaction. The Km for NAD and the maximal velocity at 25 degrees C and pH 8.0 in the presence of both compounds are 55 micron and 1,400 nmol/min/mg, respectively. The sedimentation coefficient (s020,w) of the enzyme is 5.80 S. The molecular weight is calculated to be 108,000 by sedimentation equilibrium method using a partial specific volume of 0.736 ml/g. This value is in good agreement with the molecular weight values of 115,000 and 120,000 determined by gel filtration on Sephadex G-200 and gel electrophoresis in the presence of sodium dodecyl sulfate, respectively. The enzyme is colorless and its absorption spectrum shows a maximum at 280 nm. From a CD spectrum, alpha helical content is estimated to be approximately 30%. The enzyme is a basic protein having a pI value of 9.8 and is rich in lysine rather than arginine. Neutral sugar, phospholipid, and DNA are not detected in the final preparation. These data indicate that the purified enzyme is a simple globular protein composed of a single polypeptide having an approximate molecular weight of 110,000.     

Carbon monoxide:methylene blue oxidoreductase from Pseudomonas carboxydovorans.

The enzyme carbon monoxide:methylene blue oxidoreductase from CO autotrophically grown cells of Pseudomonas carboxydovorans strain OM5, was purified to homogeneity. The enzyme was obtained in 26% yield and was purified 36-fold. The enzyme was stable for at least 6 days, had a molecular weight of 230,000, gave a single protein and activity band on polyacrylamide gel electrophoresis, and was homogeneous by the criterion of sedimentation equilibrium. Sodium dodecyl sulfate gel electrophoresis revealed a single band of molecular weight 107,000. Carbon monoxide:methylene blue oxidoreductase did not catalyze reduction of pyridine or flavin nucleotides but catalyzed the oxidation of CO to CO2 in the presence of methylene blue, thionine, toluylene blue, dichlorophenolindophenol, or pyocyanine under strictly anaerobic conditions. The visible spectrum revealed maxima at 405 and 470 nm. The millimolar extinction coefficients were 43.9 (405 nm) and 395.5 (275 nm), respectively. Absorption at 470 nm decreased in the presence of dithionite, and the spectrum was not affected by the substrate CO. Maximum reaction rates were found at pH 7.0 and 63 degrees C; temperature dependence followed the Arrhenius equation, with an activation energy (delta H degree) of 36.8 kJ/mol (8.8 kcal/mol). The apparent Km was 53 microM for CO. The purified enzyme was incapable of oxidizing methane, methanol, or formaldehyde in the presence of methylene blue as electron acceptor.     

Purification and properties of Escherichia coli dimethyl sulfoxide reductase, an iron-sulfur molybdoenzyme with broad substrate specificity.

Dimethyl sulfoxide reductase, a terminal electron transfer enzyme, was purified from anaerobically grown Escherichia coli harboring a plasmid which codes for dimethyl sulfoxide reductase. The enzyme was purified to greater than 90% homogeneity from cell envelopes by a three-step purification procedure involving extraction with the detergent Triton X-100, chromatofocusing, and DEAE ion-exchange chromatography. The purified enzyme was composed of three subunits with molecular weights of 82,600, 23,600, and 22,700 as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was determined by gel electrophoresis to be 155,000. The purified enzyme contained 7.5 atoms of iron and 0.34 atom of molybdenum per mol of enzyme. The presence of molybdopterin cofactor in dimethyl sulfoxide reductase was identified by reconstitution of cofactor-deficient NADPH nitrate reductase activity from Neurospora crassa nit-I mutant and by UV absorption and fluorescence emission spectra. The enzyme displayed a very broad substrate specificity, reducing various N-oxide and sulfoxide compounds as well as chlorate and hydroxylamine.     

Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 +/- 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine X HCl concentrations is characterized by a two-step profile: at 0.4-0.8 M, partial unfolding is parallelled by inactivation; at 2.0-2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4-1.8 M guanidine X HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine X HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine X HCl, as monitored by the decrease of protein fluorescence, is fast (less than 8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (less than 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by tau = 29 +/- 3 min (20 degrees C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate-limiting for reactivation after long-term denaturation, are characterized by a high energy of activation (112 +/- 8 kJ/mol). The slow reactivation step is compatible in rate with the first-order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62-90].     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

Bovine alpha-fetoprotein. Isolation and characterization.

Bovine AFP was purified by ion exchange chromatograph on C.M. cellulose and DEAE Sephadex A-50, gel filtration and immunosorbent technique. AFP was homogeneous when studied by gel electrophoresis under non denaturing and denaturing conditions, by ultracentrifugation and by immunological methods. The following molecular data were obtained: 1. Sedimentation equilibrium indicated a molecular weight of 66,500 and sedimentation velocity gave s degrees 20, w = 4.71 S. A partial specific volume v = 0.737 ml g-1 was derived from density measurements. 2. From these data, a Stokes radius of 3.26 nm, a diffusion coefficient D20 w = 6.61 10(-7) cm2 sec-1 and a frictional ratio f/fo = 1.21 were calculated. 2. Sodium dodecylsulphate disc electrophoresis suggests a molecular weight of 67,000. 3. Gel filtration pointed to a molecular weight of 75,000. 4. Microheterogeneity of AFP was demonstrated by isoelectric focusing. The isoelectric point of the major component is 4.6. 5. The chemical composition was determined. AFP is a glycoprotein containing 7 per cent carbohydrate including 1.67 per cent hexoses, 2.38 per cent N-acetyl glucosamine and 1.8 per cent N-acetyl neuraminic acid.     

Mechanism of hypoglycaemic action of methylenecyclopropylglycine.

Cytochrome b-245 from neutrophil plasma membranes contains two types of subunit with apparent molecular masses from gel electrophoresis in the presence of SDS of 23 kDa and 76-92 kDa. Radiation-inactivation analysis revealed a single-exponential decay process for the visible absorption of the haem chromophore in the membrane, corresponding to a molecular mass of 21 +/- 5 kDa for the haem-containing polypeptide chain. Sedimentation equilibrium of the cytochrome solubilized by the detergent Triton N101 showed that the protein was polydisperse, with a molecular mass of approx. 350 kDa for the smallest detectable species. In another detergent, n-octyl beta-O-glucopyranoside (octyl glucoside), the molecular mass of the haem-containing particle was found to be 20-30 kDa. Thus the quaternary structure of the protein breaks down in this detergent. The haem group is inferred to be attached to the smaller subunit.