Enzymic and physicochemical characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from diploid and tetraploid cultivars of perennial ryegrass

Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were isolated from several diploid and tetraploid cultivars of perennial ryegrass (Lolium perenne L.) by three different purification protocols. The apparent K_m values for substrate CO₂ were essentially identical for the fully $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$ activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and small subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report (M. K. Garrett, 1978, Nature (London)274, 913–915), these results indicate that increased ploidy level (i.e., nuclear gene dosage) has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/ oxygenase in perennial ryegrass.     

Differences in the physical properties of collagenases isolated from rheumatoid synovium and human skin

During purification of human collagenase from normal skin and rheumatoid synovium differences have been observed with regard to the size and charge properties of the two enzymes. Gel filtration experiments in Sephadex G-100 superfine have allowed molecular weights of approximately 63,000 and 32,000 daltons to be calculated for the skin and rheumatoid synovial enzyme respectively. Ion exchange chromatography using Sephadex QAE, A-50 has shown the rheumatoid synovial enzyme to possess charge properties more basic than that of the skin enzyme. Elution of collagenase activity from $\text{7}\text{1}\text{2}\text{\%}$ polyacrylamide gels has produced no evidence for a ‘fast-moving’ component of either enzyme.     

Alkali-labile oligosaccharides from bovine milk fat globule membrane clycoprotein

Phenol extraction of bovine milk fat globule membrane gave a glycoprotein fraction which, in sodium dodecyl sulphate electrophoresis, showed three major bands, all staining for both protein and carbohydrate. Alkaline borohydride treatment and desialylation of the glycoprotein fraction released the reduced disaccharide β-d-galactosyl(1 → 3)-$\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ (T-antigen), which was identified by gas chromatography using a standard. All of the disaccharide units in the native glycoprotein were shown to be substituted by sialic acid, and a tetrasaccharide containing the disaccharide plus two molecules of sialic acid was isolated following alkaline borohydride treatment of the glycoprotein and gel filtration. Periodate oxidation of native and desialylated glycoprotein, together with paper chromatography of alkali degraded oligosaccharide fragments, indicated that the major alkali-labile oligosaccharide of the glycoprotein fraction is a tetrasaccharide containing $\text{β-}\text{d}\text{-galactosyl(1 → 3)}\text{-N-}\text{acetyl-}\text{d}\text{-galactosamine}$ substituted by sialic acid at position C3 of the galactosyl and position C6 of the $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ residue. Evidence was also obtained for the presence of small amounts of unsubstituted alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ linked directly to protein in the native glycoprotein.Serological evidence using agglutinins from Vicia graminea, Arachis hypogoea and human anti-T serum confirmed the presence in the native glycoprotein of a sialic acid substituted T-antigen. Similar evidence using agglutinins from Helix pomatia and Cepaea hortensis also confirmed the presence of terminal alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ in the native glycoprotein.     

Isolation of a mitochondrial DNA topoisomerase from human leukemia cells

Mitochondria from human acute lymphoblastic leukemia cells contain an ATP-independent DNA topoisomerase which can relax negative and positive supercoils. This enzyme has been purified 200-fold by carboxymethyl-cellulose or double stranded DNA-cellulose chromatography. In contrast to the molecular weights reported for mitochondrial topoisomerases in other systems, the native leukemia enzyme has a molecular weight of 132,000 daltons as determined by gel permeation chromatography in buffer containing 0.4 M KCl. It also exhibits a sedimentation coefficient of 7.1 S when centrifuged through a 10–30% glycerol gradient in this high salt buffer. The enzyme is presumably a type I topoisomerase analogous to those found in rat liver and $\text{Xenopus}$$\text{laevis}$ mitochondria.     

Molecular weight and symmetry of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The molecular weight and polypeptide chain stoichiometry of the native pyruvate dehydrogenase multienzyme complex from Escherichia coli were determined by independent techniques. The translational diffusion coefficient (D^o_20,w) of the complex was measured by laser light intensity fluctuation spectroscopy and found to be 0.90 (±0.02) × 10^?11m²/s. When this was combined in the Svedberg equation with the measured sedimentation coefficient (s^o_20,w = 60.2 (±0.4) S) and partial specific volume ($\text{v}\text{?}\text{= 0.735 (±0.01)}\text{ml/g}$), the molecular weight of the intact native complex was calculated to be 6.1 (±0.3) × 10⁶. The polypeptide chain stoichiometry (pyruvate decarboxylase: lipoate acetyltransferase: lipoamide dehydrogenase) of the same sample of pyruvate dehydrogenase complex was measured by the radioamidination technique of Bates et al. (1975) and found to be 1.56:1.0:0.78.From this stoichiometry and the published polypeptide chain molecular weights estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, a minimum chemical molecular weight of 283,000 was calculated. This structure must therefore be repeated approximately 22 times to make up the native complex, a number which is in good agreement with the expected repeat of 24 times if the lipoate acetyltransferase core component has octahedral symmetry. It is consistent with what appears in the electron microscope to be trimer-clustering of the lipoate acetyltransferase chains at the corners of a cube. It rules out any structure based on 16 lipoate acetyltransferase chains comprising the enzyme core.The preparation of pyruvate dehydrogenase complex was polydisperse: in addition to the major component, two minor components with sedimentation coefficients (s^o_20,w) of 90.3 (±0.9) S and 19.8 (±0.3) S were observed. Together they comprised about 17% of the total protein in the enzyme sample. Both were in slowly reversible equilibrium with the major 60.2 S component but appeared to be enzymically active in the whole complex reaction. The faster-sedimenting species is probably a dimer of the complex, whereas the slower-sedimenting species has the properties of an incomplete aggregate of the component enzymes of the complex based on a trimer of the lipoate acetyltransferase chain.     

Studies on the molecular weight of the adenovirus type 2 hexon and its subunit

The molecular weight of the adenovirus type 2 hexon was calculated from sedimentation equilibrium, light scattering and sedimentation and diffusion experiments. The extinction coefficient, E_{1 cm}^1%, was determined to be 14.3 at 279 nm, from quantitative nitrogen and carbon analyses combined with the N,C content calculated from the amino acid composition. Other parameters determined were: the partial specific volume, $\text{}\text{?}\text{gn = 0.738}\text{cm}{}^3\text{g}{}^{\mathrm{?1}}$; the refractive index increment, $\text{(}\text{?n}\text{?c}\text{)}{}_{\mathrm{<mtext>T,P</mtext><mtext>,\mu </mtext>}}\text{= 0.193}\text{cm}{}^3\text{g}{}^{\mathrm{?1}}$ at 435.8 nm; the sedimentation coefficient, s_20,w⁰ = 13.0 S; and the diffusion constant, D_{20, w}⁰ = 3.32 × 10^?7 cm² s^?1. All molecular weights were between 355,000 and 363,000. Crystal density measurements were made on native and glutaraldehyde cross-linked crystals and the molecular weights calculated from these data were compared with the precise molecular weight determined by physico-chemical methods.Only one polypeptide of molecular weight 120,000 was found in reduced, or reduced and alkylated, hexon. Four or six organomercurial molecules were bound per 120,000 molecular weight of native hexon upon titration with 2-chloromercuri-4-nitrophenol and 2-chloromercuri-4,6-dinitrophenol, respectively. With 5,5′-dithiobis (2-nitrobenzoic acid) only one SH-group per 120,000 could be titrated in native hexon, but after denaturation in 1% sodium dodeeyl sulphate five more SH-groups reacted per 120,000 molecular weight. Thus there are three identical polypeptides of molecular weight 120,000 per hexon of total molecular weight 360,000.     

Isolation and properties of somatic and testicular cytochromes c from rat tissues

Somatic (c_s) and a testis-specific (c_{t I}) cytochromes c were purified to homogeneity from rat tissues (heart, liver, kidney, and testis). The purification procedure involved (1) homogenization of tissues at pH 4.5, (2) treatment with methanol-chloroform solvents, (3) hydroxylapatite column chromatography, (4) carboxymethyl-cellulose column chromatography, and (5) Sephacryl S-200 gel filtration. The isolated cytochromes c were free from polymeric and other “modified” forms, and did not bind CO, azide, or cyanide. The absorption maxima and the molecular weights of both cytochromes c_s and c_{t I} were identical. The ratio of $\text{A}{}_{\mathrm{<mtext>549.5\; </mtext><mtext>nm</mtext>}}\text{(}\text{reduced}\text{)}\text{A}{}_{\mathrm{<mtext>280\; </mtext><mtext>nm</mtext>}}\text{(}\text{oxidized}\text{)}$ for cytochromes c_s averaged 1.28. The unique properties of cytochrome c_{t I}, compared to somatic cytochrome c, were as follows: (1) different elution profiles from hydroxylapatite and carboxymethyl-cellulose column chromatography experiments, (2) less basic intrinsic molecular charge shown by the slow mobility in native polyacrylamide gel electrophoresis, (3) probable asymmetric molecular shape as evidenced from gel filtration experiments, (4) significantly higher millimolar extinction coefficient values (33.6 at 549.5 nm), (5) a low ratio (1.04) of $\text{A}{}_{\mathrm{<mtext>549.5\; </mtext><mtext>nm</mtext>}}\text{(}\text{reduced}\text{)}\text{A}{}_{\mathrm{<mtext>280\; </mtext><mtext>nm</mtext>}}\text{(}\text{oxidized}\text{)}$, and (6) difference of about 20 amino acid residues per mole.     

Evidence for an allosteric control of the pentose phosphate pathway in Candida utilis

Glucose 6-phosphate dehydrogenase from $\text{Candida utilis}$ exists in two interconvertible species having molecular weights of 110,000 and 220,000, respectively. By use of sodium dodecyl-sulfate the enzyme can be dissociated into 2 electrophoretically separable subunits. The native enzyme was shown to be strongly inhibited by low concentrations of erythrose 4-phosphate and glyceraldehyde 3-phosphate, respectively. Since both these sugar phosphates are metabolites in the later course of the oxidative pathway this regulation may be considered as a negative feedback control of the pentose phosphate cycle.     

Structural polypeptides of primate derived type C RNA tumor viruses

Proteins of gibbon ape lymphosarcoma virus (GaLV) and woolly monkey sarcoma virus, type 1, together with its associated virus ($\text{SSV-1}\text{SSAV-1}$) were analyzed by guanidine-agarose chromatography and the separation patterns were compared with those of mouse and feline type C viruses. GaLV contained five major proteins, including two glycoproteins, whereas lower mammalian viruses contained six major proteins, including two glycoproteins. The molecular weights of the five GaLV proteins closely resembled the molecular weights of the five equivalent lower mammalian viral proteins. $\text{SSV-1}\text{SSAV-1}$ showed a separation pattern similar to GaLV except it contained a low but detectable amount of an additional glycoprotein. Both GaLV and $\text{SSV-1}\text{SSAV-1}$ were deficient in a protein of molecular weight about 15,000 daltons which is found in all known type C viruses of avian, reptilian and lower mammalian species.     

Purification and characterization of insect cathepsin D

We purified a proteolytic enzyme from pupae of the blowfly Aldrichina grahami. The purification procedure consisted of fractionation with ammonium sulfate, ethanol treatment, affinity chromatography on Con $\text{A-}\text{Sepharose}$, and ion exchange chromatography on CM-Sepharose CL-6B. The purified enzyme was very labile. The lability was reduced by the use of MES buffer containing 10% ethanol, which enabled us to purify the enzyme to homogeneity. The lower the polarity of the alcohol added, the more stable the enzyme became. The enzyme had a molecular weight of 41,000, an optimum pH of 3.5, high susceptibility to pepstatin and two pI forms of 5.4 and 6.2. This enzyme resembled cathepsin $\text{D}$, a lysosomal proteinase.     

Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin

Porcine tissue plasminogen activator has been purified from delipidized heart tissue by affinity adsorption to fibrin. A crude fraction is prepared from an acid tissue extract by precipitation with ammonium sulphate. The tissue activator of this fraction is isolated by adsorption on fibrin and elution with KSCN. The procedure also includes chromatography on arginine-Sepharose and two gel-filtration steps. The final product has a specific activity of 250 000 IU/mg (±16 000) as compared to an international urokinase reference preparation. The yield calculated from the active ammonium sulphate precipitate is about 28%. An approx. 7 000-fold increase of specific activity is obtained, most of which is achieved in the fibrin step. The native tissue plasminogen activator consists of a single chain molecule with a molecular weight of 64 000 as measured by SDS-polyacrylamide gel electrophoresis. In a previous report, it was claimed that the activator is composed of two disulphide-connected polypeptide chains. These results were due to a preparation artefact, caused by proteolytic activity present in the tissue extracts. The introduction of the protease inhibitor aprotinin and 6-amino-hexanoic acid in the purification procedure has abolished the effect of the protease contaminant, leading to the production of a one-chain activator. Treatment with plasmin transforms the native, one-chain tissue activator into a variant composed of two chains of about equal size (M_r 32 000) connected by disulphide bonding. This modified activator is indistinguishable from the one obtained at insufficient protection against proteolytic enzymes. The cleavage by plasmin causes about an 8-fold increase of amidolytic activity as measured on $\text{H}\text{-}\text{D}\text{-}\text{Val}\text{-}\text{Gly}\text{-}\text{Arg}\text{-p-}\text{nitroanalide}$. The fibrinolytic activity as measured by clot lysis is only slightly increased. The physiological significance of the cleavage is discussed.     

Direct fluorometric analysis of benzo(a)pyrene metabolite formation by mouse liver microsomes

We have examined the metabolites produced by in vitro incubation of benzo(a)pyrene with 3-methylcholanthrene-induced mice liver microsomes. Our objective was to observe directly a possible difference in microsomal enzyme systems of animal models having different susceptibility to chemical carcinogens. The metabolites produced by the two animal models,$\text{C57BL}\text{6J}$ and $\text{DBA}\text{2}$ mice, were analyzed by a highly sensitive, “three-dimensional” fluorescence plotting technique. The fluorescence spectra of the total ethyl acetate-soluble metabolites clearly indicate that the metabolites produced by $\text{DBA}\text{2}$ enzymes were predominantly monohydroxylated benzo(a)pyrene while those produced by the liver microsomes of $\text{C57BL}\text{6J}$ were highly enriched with the 7,8-dihydrodihydroxybenzo(a)pyrene type.     

Equilibrium centrifugation of proteins in acidic solutions. Beta-lactoglobulin A in aqueous acetic, propionic, and butyric acids.

Short chain aliphatic acids are almost neutrally buoyant in aqueous solutions, and preferential interaction of macromolecules with these solvent components should not greatly affect apparent molecular weights determined by equilibrium ultracentrifugation. The feasibility of molecular weight estimations using native, neutral pH values of partial specific volume has been tested: equilibrium ultracentrifugation of β-lactoglobulin A (β-LgA) has been carried out in aqueous acetic, propionic, and butyric acids in the absence of any other added electrolyte. These solutions are highly nonideal because of the extreme Donnan effect. Apparent molecular weights estimated at infinite dilution using the native neutral pH value of the partial specific volume, $\text{v}{}_{\mathrm{p}}$, differed by less than 5% from the monomer formula weight. The 10 m acids appear to be least effective as dissociating agents for β-LgA, with a weak reversible monomer-dimer association suggested in 10 m acetic acid, with significant heterogeneity apparent in 10 m propionic acid, and with a lack of direct solubility in 10 m butyric acid. All the 0.1 m acids and all the 1 m acids were essentially equally effective as dissociating agents, with the exception of 1 m butyric acid which dissolved β-LgA only slowly to give significantly heterogeneous solutions. From these results and from our previous experiments with aldolase (6), it appears feasible to use the native values of $\text{v}{}_{\mathrm{p}}$ to obtain estimates of molecular weights of proteins in aqueous organic acids as dissociating agents.     

Purification and characterization of a lysozome from wheat germ

Several proteins of wheat germ were able to lyse Micrococcus luteus cells. One lysozyme, named W1A, was purified by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and preparative polyacrylamide gel electrophoresis (PAGE) under native conditions. The enzyme had a molecular weight of 25 400 as determined by sodium dodecyl sulfate (SDS)-PAGE. The reducing groups released from the lysis of Micrococcus cell walls by W1A lysozyme were $\text{N-}\text{acetylmuramic}$ acid residues as for hen egg white lysozyme (HEWL). Chitin substrates were hydrolyzed to some extent by this enzyme. With Micrococcus cells as substrate, the pH optimum for W1A lysozyme was 6.0 at an optimal ionic strength of 0.05. Under these conditions, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 166 mg/l with purified Micrococcus cell walls and the V_max value was 0.56 A₅₄₀ unit/min at 22°C. W1A lysozyme exhibited the highest lytic activity at 60°C whereas the enzyme was inactive above 90°C. W1A lysozyme was strongly inhibited by poly-l-lysine and glycol chitosan. This is the first report of the presence of multiple electrophoretic forms of plant lysozyme activity as determined by native PAGE.     

Preparation and immunochemical properties of methoxypolyethylene glycol-coupled and N-carboxymethylated derivatives of ragweed pollen allergen, antigen E.

A methoxypolyethylene glycol (PEG)-coupled and several N-carboxymethylated (N-CM) derivatives of antigen E, the major allergenic protein of ragweed pollen, were prepared. The PEG derivative contained seven residues of PEG groups (residue weight about 2100) per molecule of protein and the groups were linked to the lysyl residues of antigen via the 2,6-positions of 4-hydroxy-triazine nucleus. The maximally N-CM derivative contained, respectively, 10, 6, and 2 residues of mono-CM, di-CM, and unmodified lysyl residues per molecule of protein. The CM groups were introduced reductively on reaction with glyoxylic and sodium cyanoborohydride and the extent of mono- and dicarboxymethylation was controlled more by the concentration of cyanoborohydride than by that of glyoxylic acid. The molar allergenic activities of the PEG and the N-CM derivatives in man were, respectively, 0.02 and 0.5 of that of the native antigen. Rabbits immunized with the PEG derivative gave antibody titers about $\text{1}\text{8}$th of those obtained with animals immunized with the native antigen. However, the rabbits preimmunized with the PEG derivative gave a vigorous secondary response on challenge with the native antigen and their titers approached those of rabbits preimmunized with the native antigen. The immunogenicity of the reduced and S-carboxymethylated derivative of antigen E which has the denatured conformation was studied as a control. Rabbits immunized with the S-CM derivative gave antibody titers $\text{1}\text{34}$th of those obtained with animals immunized with the native antigen; on secondary challenge with the native antigen, these rabbits gave antibody titers about $\text{1}\text{6}$th of those of animals preimmunized with the native antigen.     

A manganese-stimulated endonuclease from Bacillus subtilis

An endonuclease activity has been identified in extracts of $\text{Bacillus subtilis}$. This activity is stimulated by Mn⁺⁺ or Ca⁺⁺ ions but not by Mg⁺⁺ ions. The enzyme catalyzes the breakdown of native DNA of high molecular weight to fragments of molecular weights ranging from 3 × 10⁶ to 20 × 10⁶. A variety of DNA's from sources such as $\text{B. subtilis, Salmonella}$ and T7 phage are attacked. About 61% of the activity of the cells is released into the medium during protoplast formation under conditions where 98% of the glucose 6-P dehydrogenase activity is retained by the cells.     

Examination and rapid analysis of hepatic cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using gel-permeation high performance liquid chromatography

Gel-permeation high performance liquid chromatography was used to examine the molecular characteristics of the cytosolic and nuclear receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin. The physical properties for this receptor previously established by sucrose-density gradient analysis have been maintained by the technique. The high performance liquid chromatography method offers the advantages of rapid separation and reproducibility thus minimizing possible effects of proteolytic enzymes or dissociation of the ligand-receptor complex. The specific binding of $\text{[}{}^3\text{H}\text{]2,3,7,8-}\text{tetrachlorodibenzo}\text{-p-}\text{dioxin}$ as measured by this system also corresponds quantitatively to that determined by the hydroxyapatite method. These methods will facilitate a further understanding of the biochemical actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds, as well as an understanding of the physiological function of this receptor molecule.     

Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography

Toluene dioxygenase, from $\text{Pseudomonas}\text{putida}$, oxidizes toluene to (+)-$\text{cis}$-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxin_TOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the $\text{Pseudomonas}$ reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISP_TOL.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.