Rat liver uroporphyrinogen III synthase has similar properties to the enzyme from Euglena gracilis, including absence of a requirement for a reversibly bound cofactor for activity.

Uroporphyrinogen III synthase purified from rat liver is a monomer of Mr 36,000 by gel filtration and 28,000 by SDS/polyacrylamide-gel electrophoresis. The enzyme exists in two interconvertible forms separable on h.p.l.c. Both forms of the enzyme could be renatured with full activity after SDS/polyacrylamide-gel electrophoresis, demonstrating the absence of a reversibly bound cofactor. The enzyme activity could be inhibited by pyridoxal 5'-phosphate in the absence and in the presence of NaBH4, consistent with (an) essential lysine residue(s). The enzyme thus shows great similarity to that from Euglena gracilis.     

Comparison of tRNA nucleotidyltransferase from Escherichia coli and Lactobacillus acidophilus.

Purification of tRNa nucleotidyltransferase from Lactobacillus acidophilus ATCC 4963 and Escherichia coli MRE 600 by preparative polyacrylamide gel electrophoresis is described. Both enzymes gave a single band on analytical polyacrylamide-gel electroesis and sodium dodecylsulfate gels. Chromatography of the high speed supernatant from Lactobacillus at low salt concentrations gave three enzyme fractions of molecular weights about 45 000, 90 000, and 120 000. At 1M NaCl only the first enzyme fraction was found. Kinetic data for both enzymes are given.     

Protein disulphide-isomerase of chick-embryo tendon.

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.     

Isolation and renaturation of alpha 2-macroglobulin receptor from diploid human fibroblasts.

alpha 2-Macroglobulin receptor was extracted from human diploid fibroblasts and purified by affinity chromatography in a single step. The receptor had mol.wt. 125 000 after sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. The isolated receptor was separated by SDS/polyacrylamide-gel electrophoresis, transferred on to nitrocellulose sheets and subsequently renatured, as shown by a specific binding test, by incubation with Nonidet P40.     

Solubilization and purification of rat liver 5''-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent.

1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.     

Affinity-chromatographic purification of S-adenosyl-L-homocysteine hydrolase. Some properties of the enzyme from rat liver.

S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose. The purified enzyme was free from adenosine kinase and adenosine deaminase activities and was homogeneous on SDS/polyacrylamide-gel electrophoresis which gave a subunit mol.wt. of 47 000. The native enzyme showed some microheterogeneity on polyacrylamide-gel electrophoresis under increased-resolution conditions but was homogeneous on isoelectric focusing (pI 5.6). The molecular weight of the native enzyme was about 220 000 as judged by pore-gradient electrophoresis. The native enzyme bound adenosine tightly and showed Km values of 0.6 microM, 0.9 microM and 60 microM for adenosine, S-adenosyl-L-homocysteine and L-homocysteine respectively. The enzyme was rapidly inactivated when incubated in the presence of adenosine, S-adenosyl-L-homocysteine or several adenosine derivatives or analogues. Inactivation took place both at 0 and 37 degrees C. Freezing in the absence of glycerol resulted in the appearance of dissociation products of the oligomeric protein. Multimer formation was observed at low thiol concentrations.     

Purification of the enzyme NADPH: protochlorophyllide oxidoreductase.

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.     

Soluble adenylate cyclase activity in Neurospora crassa.

A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.     

Purification and characterization of rat liver glycosylasparaginase.

1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.     

Lens neutral endopeptidase occurs in other bovine and human tissues.

Lens neutral endopeptidase (EC 3.4.24.5) was previously thought to be unique to the eye lens. We report here the finding of a neutral endopeptidase, in a variety of bovine and human tissues, which is very similar both biochemically and immunologically to the lens endopeptidase. SDS/polyacrylamide-gel electrophoresis of partially purified enzyme fractions from various bovine tissues shows the characteristic pattern of at least eight bands with Mr values ranging from 24,000 to 32,000 which was described for the bovine-lens neutral endopeptidase. The relative activity of the enzyme varies from tissue to tissue with lung having the highest activity. Partially purified enzyme fractions from these tissues cross-react with antiserum raised in rabbit against bovine lens endopeptidase showing apparent identity when examined side by side in Ouchterlony double-diffusion tests. The human enzyme also cross-reacts with the antiserum but when tested by double-diffusion against the bovine enzyme the precipitin lines show spurring at the joining edges indicating a structural difference between the human and the bovine enzymes. It was also found by Western blot experiments, after denaturing polyacrylamide-gel electrophoresis of the enzyme, that the polypeptide components of the human and bovine enzymes show somewhat different banding patterns.     

Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli.

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.     

Isolation of the smallest component of silk protein.

Silk proteins were solubilized from cocoons with ethylenediamine/cupric hydroxide solution. A series of polymers of the smallest component, detected by polyacrylamide-gel electrophoresis, could be converted into the smallest component by reduction and aminoethylation. Fibroin and sericin fractions were separated by precipitation of sericin at pH 5.5. On gel electrophoresis, sericin showed distinct bands but fibroin did not. The components of fibroin and sericin were fractionated by gel filtration on Sepharose 6B. The smallest component in the sericin fraction was purified by rechromatography and showed a single band on gel electrophoresis. Its mol. wt. was 24 000, and its amino acid composition was determined.     

Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens.

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Purification and properties of the high-activity form of the enzyme.

1. The high-activity form of aminolaevulinate synthetase has been prepared from extracts of semi-anaerobically grown cells of Rhodopseudomonas spheroides, which were allowed to become activated in air. Specific activity was 130 000--170 000 nmol of aminolaevulinate/h per mg of protein at 37 degree C. 2. Enzyme fraction Ia prepared on DEAE-Sephadex was a mixture of four active enzymes, pI5.55, 5.45, 5.35 and 5.2, when prepared in either Tris or phosphate buffers and when extracts were activated by air or by cystine trisulphide. 3. The enzyme was further purified by preparative polyacrylamide-gel electrophoresis in imidazole/veronal buffer, pH 7.6, followed by gel filtration on Sephadex G-100 and concentration with DEAE-Sephadex. 4. The most active enzyme, pI 5.55, ran as a single protein band, mol.wt. 49 000, in sodium dodecyl sulphate and 2-mercaptoethanol. The apparent molecular weight under non-denaturing conditions was 62 000--68 000 on Sephadex G-100 or G-200, pH 7.5, and on polyacrylamide-gel electrophoresis, pH 8.5, at enzyme concentrations below 10 000 units/ml, i.e. less than 60 microgram of protein/ml, and the enzyme was mainly monomeric. 5. The enzyme was homogeneous by gel disc electrophoresis at pH 8.9 and 7.6, but a slightly more diffuse band of protein was obtained during electrophoresis in glycine buffer, pH 7.4. 6. Enzyme samples possessed an intrinsic yellow fluorescence when viewed under u.v. light and this fluorescence coincided exactly with enzymic activity on gel electrophoresis. Fluorescence maxima were 420 nm (excitation) and 495 nm (emission). 7. Radioactive 35S-labelled enzyme had 14 atoms of sulphur/mol of protein (or/40 leucine residues) of which 5--6 residues were cyst(e)ine and 8--9 residues were methionine. 8. Mo carbohydrate was detected apart from glucose, which prevented accurate determination of tryptophan with methanesulphonic acid and tryptamine.     

Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles.

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.     

Trypanosoma cruzi adenylate cyclase activity. Purification and characterization.

Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.     

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase.

A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.     

Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate.

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.     

The purification and characterization of 3-dehydroquinase from Streptomyces coelicolor.

The enzyme 3-dehydroquinase was purified over 4000-fold to homogeneity from Streptomyces coelicolor. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of SDS was 16,000. The native Mr estimated by gel filtration on a Superose 6 column was 209,000, indicating that the enzyme is a large oligomer. The enzyme was found to be extremely thermostable. This stability, along with the structural and kinetic properties of the enzyme, suggest that it is very similar to the quinate-inducible 3-dehydroquinase found in Neurospora crassa and Aspergillus nidulans. This similarity was confirmed by direct N-terminal sequencing.     

Cathepsin D from pig myometrium. Characterization of the proteinase.

The purification of cathepsin D from pig uterus by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose was described previously [Afting & Becker (1981) Biochem. J. 197, 519-522]. In this paper, chemical and physical properties of the proteinase are presented. The purified enzyme showed three bands on SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis, one main band corresponding to an Mr of 31 000 and two minor bands with Mr values of 43 000 and 15 000 respectively. Gel filtration on Bio-gel P-150 and sedimentation-diffusion equilibrium studies give an Mr for the main band of about 35 000. The pI of the enzyme was determined to be 7.2. Haemoglobin was the best substrate, with a Km value of 6.4 X 10(-6)M. It was hydrolysed with a pH optimum between 3.0 and 3.3 for a substrate concentration of 100 microM. The proteinase was stable over the pH range of 3.5-6.5. At pH 6 the enzyme showed stability up to a temperature of 50 degrees C; at pH 3 the activity was already decreased below 40 degrees C. Carbohydrate studies resulted in the staining of all three bands on an SDS/polyacrylamide gel by thymol/H2SO4. After treatment with endo-beta-N-acetylglucosaminidase H, all three bands were shifted to a region of lower Mr. Of various inhibitors tested, only pepstatin was strongly inhibiting, with a Ki of 2.1 X 10(-9)M.