Reactivation of the EDTA-treated arginase from rat and calf liver.

1. The anionic calf liver arginase, like the cationic rat liver enzyme, is inactivated by EDTA-treatment. The activity is fully restored by Mn2+. A smaller effect is observed with Cd2+, Ni2+ and Co2+. 2. The EDTA-inactivated calf liver arginase, unlike the rat liver enzyme, does not dissociate into subunits, and its mol.wt. (120 000) is unchanged. 3. The reactivation of rat liver arginase subunits (mol.wt. 30 000) by Ni2+ is accompanied, similarly as in the case of Mn2+, by reassociation to the form of mol.wt. 120 000, i.e. the same as for the native enzyme. 4. It is suggested that Mn2+ in arginase is bound at the active site and at the site responsible for maintenance of the oligomeric structure. In calf liver enzyme this binding site is inaccessible to the chelating agent.     

Purification and some properties of nitrate reductase (EC 1.7.99.4) from Escherichia coli K12.

1. Nitrate reductase was purified 134-fold from Escherichia coli K12. The purification procedure involves the release by Triton X-100 of the enzyme from the cell envelope. i. The purified enzyme exists in aqueous solution either as a monomer (mol. wt. about 220 000) or as an associated form (probably a tetramer; mol.wt. about 880 000). 3. The purified enzyme has three subunits with apparent mol.wts. of 150 000, 67000 and 65000. An additional subunit of apparent mol.wt. 20000 is present in a haem-containing fraction that is also produced by the preparative procedure described. 4. None of the enzyme subunits is present in the cell envelope of cells grown in the absence of nitrate. 5. Reversible changes in the activity of nitrate reductase in vitro with FMNH2 as reductant can be induced under circumstances which are without effect on the reduced Benzyl Viologen-NO3-activity.     

The structure of L-lactate oxidase from Mycobacterium smegmatis.

1. An improved purification was developed for L-lactate oxidase from Mycobacterium smegmatis. 2. The mol.wt. of the native enzyme by a sedimentation-equilibrium analysis was 345 000, and other ultracentrifuge methods gave values in the range 345 000-350 000. 3. An amino acid analysis, determinations of protein and flavin, a sedimentation-velocity analysis and an approach to equilibrium analysis gave values for the subunit mol.wt. in the range 43 500-47 000. 4. It was concluded that L-lactate oxidase contains eight subunits of mol.wt. 43 500. 5. Cross-linking of the subunits with dimethyl suberimidate and electron-microscopy studies were consistent with an octameric structure.     

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.     

Purification of two hexosaminidases from human kidney.

Hexosaminidase forms A and B were isolated from human kidney in a homogeneous state as demonstrated by electrophoretic and enzymic criteria. The enzymes were stable for at least 18 months when stored at -20 degrees C in 0.025 M-phosphate buffer, pH 6.5. The molecular weights of forms A and B were estimated by gel filtration to be 111 000 +/- 1500 and 114 000 +/- 1600 respectively. The molecular weights of hexosamidase A and B subunits were determined by using polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Hexosaminidase A dissociated into one subunit with mol.wt. 68 000. Hexosaminidase B dissociated into three subunits with mol. wts. 100 000, 68 000 and 37000 respectively, and one protein band of mol.wt. 140 000. After treatment of hexosaminidases A and B with iodoacetic acid, the molecular weights of the carboxymethylated polypeptide subunits were also estimated. Carboxymethylated hexosaminidase A dissociated into one major subunit of mol.wt. 18 000 and two other protein bands of mol.wts. 65 000 and 100 000. Carboxymethylated hexosaminidase B dissociated into one major subunit for mol.wt. 19 000 and an additional band of mol.wt. 37 000. The Km of the enzymes for the synthetic substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside was 0.8 mM. Both enzymes were inhibited or activated by various metal ions. Double pH optima for the enzymes were found at pH 4.5 and 4.8.     

The tryptophanase from Proteus rettgeri, improved purification and properties of crystalline holotryptophanase.

The inducible tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) was crystallized in holoenzyme from the cell extract of Proteus rettgeri. The purification procedure included ammonium sulfate fractionation, heat treatment at 60 degrees C, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystallization was performed by the addition of ammonium sulfate to the purified enzyme solution containing 20% (v/v) glycerol, 0.1 mM pyridoxal phosphate and 10 mM mercaptoethanol. The crystallized enzyme was yellow and showed absorption maxima at 340 and 420 nm. The crystalline holotryptophanase preparation was homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight of the enzyme was calculated as approx. 222 000. The amount of pyridoxal phosphate bound to the enzyme was determined to be 4 mol per mol of the enzyme. The enzyme is composed of four subunits of identical molecular size (mol. wt 55 000) and irreversibly dissociates into these subunits in the presence of a high concentration of sodium dodecylsulfate or guanidine hydrochloride. The NH2-terminal amino acid of the enzyme was identified as alanine.     

On the subunit structure of particulate aminopeptidase from pig kidney.

Solubilization of particulate aminopeptidase (EC 3.4.11.2) from pig kidney with Triton X-100 yields an aggregate (mol. wt. approx. 10(6)) that decomposes into "free" aminopeptidase (mol. wt. 280 000) either upon autolysis at pH 5 or after exposure to trypsin. Both procedures yield free enzymes that are identical with respect to electrophoretic mobility, enzymatic activity and zinc content. After dissociation, the enzyme resulting from autolysis yields a single subunit of 140 000 molecular weight while the trypsin-treated enzyme produces three fragments (140 000, 95 000 and 48 000 mol. wt.). As the aggregate is formed by subunits 10 000 daltons heavier than those of the free enzyme, the existence of a hydrophobic portion anchoring the enzyme to the membrane might be postulated. Reactivation experiments carried out on the three purified fragments of urea-denatured aminopeptidase show that the 140 000 molecular weight subunit is the only one able to yield an active enzyme (after spontaneous dimerization). It can be concluded that the smaller fragments are artefacts resulting from trypsin degradation during purification.     

Isolation and properties of alpha-D-mannosidase from human kidney.

Alpha-D-Mannosidase activity exists in three forms that can be separated by DEAE-cellulose chromatography, alpha-D-Mannosidase was isolated from human kidney in a homogeneous state, and was purified 2100-fold, with p-nitrophenyl alpha-D-mannoside as substrate. The purified alpha-D-mannosidase was practically free from all other glycosidases tested. The Km of the synthetic substrate with the enzyme was 1 X 10(-3) M and the pH optimum 4.5. It was inhibited by heavy metals, sodium dodecyl sulphate, urea and compounds that react with the thiol groups, and was activated by Zn2+, Na+, 2-mercaptoethanol, human albumin and gamma-globulin. The mol. wt. of the enzyme was estimated to be 180 000 +/- 4500. After pretreatment with 2-mercaptoethanol and sodium dodecyl sulphate, alpha-D-mannosidase dissociated into subunits of mol. wts. of 58 000 +/- 600 and 30 000 +/- 380 respectively. Subunits of the same molecular weights were also obtained after the enzyme was heated at 100 degrees C.     

Properties and subunit composition of RNA polymerase II from plant cell cultures.

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH4)2SO4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn2+; alpha-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.     

Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24--48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.     

The Structure of Legumin of Vicia faba L.--a Reappraisal

Two-dimensional electrophoretic studies have shown a wide rangeof heterogeneity in the subunits of legumin isolated from seedsof Vicia faba L. As many as 10 disulphide-linked subunit pairsin the mol. wt. range 37 000–79 000 have been observed.Each subunit pair separated on reduction by 2-mercaptoethanolinto a large acidic and a small basic subunit, each of whichwas shown to be heterogeneous in charge by isoelectric focusing.More heterogeneity was found in the large subunits (mol. wt.range 23 000–58 000; pl range 4.6–6.1) than in thesmall subunits (mol. wt. range 21 000–23 000; pl range8.2–8.5). Most legumin molecules seemed to be formed byrandom association of subunit pairs, although one subunit pairassociated only with itself to give a molecular type separableunder non-dissociating conditions.     

Comparative study on conformational stability and subunit interactions of two bacterial asparaginases.

The denaturation and reconstitution of Erwinia carotovora and Escherichia coli L-asparaginases has been followed by optical rotatory dispersion, circular dichroism and analytical ultracentrifugation. Denaturation in urea results in dissociation of the native enzyme (mol. wt. 140 000 approx.) to produce unfolded subunits (mol. wt. 35 000 approx.); the Erwinia L-asparaginase subunits can be refolded by dilution or dialysis in alkaline conditions, pH 10.5, without aggregation to the active tetramer, to give a rather unstable solution of a monomer possibly in equilibrium with dimer. These alkaline-reconstituted subunits undergo a conformational change to a more ordered state in the presence of sodium dodecylsulphate, similar to those produced by the action of sodium dodecylsulphate on the native enzyme. If the denatured subunits are reconstituted in the pH range 5.0-7.5, the enzymically active tetramer is reformed in up to 80% yield, depending upon the conditions of temperature and concentration. Kinetic data for these various transitions suggest that dissociation is a rate-limiting step while conformational changes of the polypeptide chains are relatively much more rapid. The possible significance of these different rates of change to therapeutic considerations is discussed.     

Electron transport systems of Candida utilis. Purification and properties of the respiratory chain-linked external NADH dehydrogenase+

The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 · 10⁶. The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ₆ and CoQ₁ derivatives as acceptors. Rotenone (10^?5 M) and seconal (10^?3 M) do not inhibit enzymatic activity.     

The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them.

The killer character of the yeast Kluyveromyces lactis is associated with the presence of the linear DNA plasmids k1 and k2 and results from the secretion of a protein toxin into the growth medium. We find that toxin activity co-purifies with three polypeptides which we have termed the alpha- (mol. wt 99,000), beta- (mol. wt 30,000) and gamma- (mol. wt 27,500) subunits. The alpha-subunit appears to contain a single asparagine-linked oligosaccharide chain but neither of the smaller subunits is glycosylated. The N-terminal amino acid sequence of each subunit has been determined. Comparison of these data with the DNA sequence of plasmid k1 indicates that it encodes all three subunits. The alpha- and beta-subunits must be processed from the primary translation product of a single gene by an enzyme related to the KEX2 endopeptidase of Saccharomyces cerevisiae.     

Purification and characterization of rat adipose tissue lipoprotein lipase.

Lipoprotein lipase (EC 3.1.1.34) extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (v/v) glycerol and 0.1% (v/v) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the partially purified enzyme preparation revealed the presence of two major Coomassie-staining bands (mol.wts. 62 000 and 56 000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]di-isopropyl fluorophosphate resulted in the incorporation of radiolabel into the band of mol.wt. 56 000, but not into the band of mol.wt. 62 000. Both the amount of the 56 000-mol.wt. polypeptide and the incorporation of [1,3-3H]di-isopropyl fluorophosphate into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats. whereas the amount of the 62 000-mol.wt. polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of mol.wt. 56 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These results suggest that the polypeptide of mol.wt. 56 000 corresponds to the subunit of lipoprotein lipase, whereas the 62 000-mol.wt. polypeptide probably represents antithrombin-III.     

Sulfite oxidase from Merluccius productus

Sulfite oxidase (sulfite:oxygen oxidoreductase, EC 1.8.3.1) was purified 482-fold from liver of the Pacific hake Merluccius productus. The molecular weight of the enzyme was found to be 120 000 by gel exclusion chromatography on Sephadex G-100. Electrophoretic analysis on sodium dodecyl sulfate (SDS)-polyacrylamide gel revealed that the enzyme was composed of two subunits whose molecular weight was estimated to be 60 000. The pH optimum of the enzyme was 8.7; Ks for sulfite, 2.5 x 10(-5) M; and that for cytochrome c, 3.6 x 10(-7) M. The enzyme elicited an EPR signal at g = 1.97 characteristic of pentavalent molybdenum. Colorimetric analysis also disclosed that the enzyme contained 2 mol each of heme and molybdenum per mol of protein. This fish liver homogenate in isotonic sucrose solution was fractionated by differential centrifugation into nuclei, mitochondria, microsomes and supernatant (100 000 X g). The major portion of sulfite oxidase activity was found in mitochondria. The sulfite oxidase activity was markedly high in liver and kidney, as compared with that in heart, spleen, muscle, gill and eye.     

Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue

A bisazido derivative was synthesized from bis(3-aminopyridinium)-1,10-decane diiodide and it was shown that it was bound (KD congruent to 2.2 muM) specifically to purified acetylcholine receptor and fulfilled the requirements for a photoaffinity label. Like the parent compound the derivative could transform membrane-bound receptor from a low ligand affinity conformation(s) to a high ligand affinity form (s), a transition which is thought to resemble desensitization processes observed in vivo. Photolysis of 3H-labeled bisazido reagent was carried out in the presence of the receptor. After dodecyl sulfate-polyacrylamide gel electrophoresis of labeled purified receptor two of the four subunits (mol wt 40 000 and 60 000) contained 90% of the bound radioactivity while for membrane-bound receptor the subunits of mol wt 40 000 and 50 000 were labeled. The results favor the assumption that the specific ligand binding sites are located on mol wt 40 000 subunits and labeling of the other subunits reflects (a) their proximity to the ligand-binding site and (b) alterations in subunit topography between membrane-bound and solubilized states.     

The subunits of human hexosaminidase A.

Previous studies of the subunit structure of hexosaminidase gave ambiguous results, but suggested that the enzyme was composed of six equally sized subunits. Dissociation of hexosaminidase A with p-chloromercuribenzoate produces an alkylated fragment with mol.wt. approx. 50000, which is converted into hexosaminidase S by treatment with dithiothreitol. Treatment of native hexosaminidase A with sodium dodecylsulphate results in the formation of a large and a small fragment. However, although the native enzyme has a sedimentation coefficient of 5.8S, dissociation by S-carboxymethylation and maleic anhydride treatment results in subunits exhibiting a single schlieren boundary on analytical ultracentrifugation with a sedimentation coefficient of 2.18S. These results indicate that the enzyme is composed of four subunits, each with molwt. approx. 25000-27000. The mol.wt. of the native enzymes is calculated to be approx. 110000. Our data are consistent with the subunit structures of hexosaminidases A, B and S as being alpha2beta2, beta4 and alpha4 respectively.     

Subunit composition and structure of subcomponent C1q of the first component of human complement.

1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.     

Purification and characterization of intestinal alkaline phosphatase from harp seal

1. Alkaline phosphatase (EC 3.1.3.1.) from harp seal (Phagophilus groenlandicus) has been purified by concanavalin A-Sepharose chromatography to homogeneity with a specific activity of 1200-1500 units/mg of protein. 2. The mol. wt of the enzyme and its subunits were estimated as 260,000 and 70,000, respectively. By chromatofocusing the isoelectric point of this enzyme is 5.5. 3. With p-nitrophenylphosphate, pH-optimum and KM for the enzyme are 9.8 and 0.9 mM, respectively. 4. The enzyme was strongly inhibited by Sn4+, Fe3+ and Zn2+, whereas Mg2+ and Mn2+ were effective activators of the enzyme. Seal alkaline phosphatase was slightly inhibited by high concentrations of Ca2+ and Cr3+. 5. The enzyme activity reached a maximum at 55-60 degrees C. It was shown that the heat stability of seal and calf intestinal alkaline phosphatases were equal at 37 and 56 degrees C.