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

1979 ◽  
Vol 181 (3) ◽  
pp. 513.b1-513.b1
Keyword(s):  
High Activity ◽  
Synthetase Activity ◽  
Purification And Properties ◽  
Activity Form ◽  
Rhodopseudomonas Spheroides

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides

1976 ◽  
Vol 273 (924) ◽  
pp. 79-98 ◽  
Keyword(s):  
High Activity ◽  
Photosynthetic Pigment ◽  
Synthetase Activity ◽  
Pigment Synthesis ◽  
Cellular Content ◽  
Rapid Fall ◽  
Activity Form ◽  
Oxygen Tensions ◽  
Rhodopseudomonas Spheroides ◽  
Low Activity

Rhodopseudomonas spheroides can grow in a defined medium with either light or oxygen as an energy source. Cells grown anaerobically or at very low oxygen tensions are rich in the photosynthetic pigment bacteriochlorophyll, whereas this pigment is virtually absent in cells grown under high oxygen tensions. Aminolaevulinate synthetase, the first enzyme on the pathway to bacteriochlorophyll, appears to play an important role in the control of bacteriochlorophyll synthesis. Thus, the enzyme has a high activity in extracts of pigmented cells and a low activity in extracts of non-pigmented cells. Further, oxygenation of a pigmented culture causes immediate cessation of pigment synthesis and produces a rapid fall in the activity of aminolaevulinate synthetase. This loss of activity appears to be due to the loss of an endogenous activator of the enzyme. Thus, pigmented cells contain cystine trisulphide, which at μM concentrations is an activator of aminolaevulinate synthetase, while oxygenation causes a rapid fall in the cellular content of this trisulphide. Cystathionase (EC 4 2.1.15) extracted from pigmented cells can catalyse the formation of cystine trisulphide from cystine, while rhodanese (EC 2.8.1.1) extracted from the same cells can catalyse the degradation of cystine trisulphide in the presence of sulphite to form cystine and thiosulphate. It is proposed that the cellular content of cystine trisulphide is controlled by changes in the levels of substrates for cystathionase and possibly rhodanese rather than changes in the amounts of these enzymes. Cystine trisulphide controls the activity of aminolaevulinate synthetase by converting a low-activity form of the enzyme (b-form) into a high-activity form (a-form). The fall in aminolaevulinate synthetase activity on oxygenation appears to be the result of cessation of conversion of b-form into a-form, along with a conversion of a-form into b-form. Factors affecting the equilibrium between the forms and the possible mechanisms for their interconversion are discussed.

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

1979 ◽  
Vol 177 (2) ◽  
pp. 649-659 ◽  
Author(s):  
R C Davies ◽  
A Neuberger
Keyword(s):  
High Activity ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Accurate Determination ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synthetase Activity ◽  
Activity Form ◽  
Rhodopseudomonas Spheroides

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.

scholarly journals Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The purification and properties of an endogenous activator of the enzyme

1973 ◽  
Vol 136 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Albert Neuberger ◽  
John D. Sandy ◽  
George H. Tait
Keyword(s):  
Pyridoxal Phosphate ◽  
Sodium Succinate ◽  
Active Form ◽  
Low Molecular Weight ◽  
Synthetase Activity ◽  
Temperature Dependent ◽  
Endogenous Factors ◽  
Ph Values ◽  
Purification And Properties ◽  
Rhodopseudomonas Spheroides

1. A low-molecular-weight activator of 5-aminolaevulinate synthetase was detected in extracts of Rhodopseudomonas spheroides. The compound activates the enzyme extracted from oxygenated semi-anaerobically grown organisms by a factor of 6–8. 2. The activator was extensively purified, but owing to the exceedingly small amounts that could be extracted in the active form its structure was not determined. 3. The activator contains an acetylatable amino group; it is more stable at acid than at alkaline pH values; it is stable to treatment with I2–KI or potassium ferricyanide, but irreversibly inactivated by Na2S2O4 or NaBH4. 4. The chromatographic, electrophoretic, chemical and stability properties of the activator are similar to those of pteridines; purified activator preparations contain pteridines, as shown by their fluorescence spectrum. This does not, however, constitute an identification of the activator. 5. The activator enhances the activity of crude and partially purified enzyme and does not appear to require other endogenous factors or a supply of air to produce activation. Activation of the purified enzyme, however, requires the presence of either pyridoxal phosphate or sodium succinate. In the absence of both these factors the activator produces a time- and temperature-dependent decay of activity.

scholarly journals Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase

1979 ◽  
Vol 177 (2) ◽  
pp. 661-671 ◽  
Author(s):  
R C Davies ◽  
A Neuberger
Keyword(s):  
Schiff Base ◽  
High Activity ◽  
Pyridoxal Phosphate ◽  
Enzymic Activity ◽  
Synthetase Activity ◽  
Dilute Solution ◽  
Concentrated Solutions ◽  
Active Centre ◽  
Rhodopseudomonas Spheroides ◽  
Low Activity

1. Pyridoxal 5′-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate–Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme–Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the “structural” pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active.

scholarly journals Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides a role for trisulphides

1975 ◽  
Vol 150 (2) ◽  
pp. 245-257 ◽  
Author(s):  
J D Sandy ◽  
R C Davies ◽  
A Neuberger
Keyword(s):  
Ion Exchange ◽  
Extraction Procedure ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Synthetase Activity ◽  
Multiple Forms ◽  
Anaerobic Culture ◽  
Structural Requirement ◽  
Rhodopseudomonas Spheroides ◽  
Low Activity

1. The aminolaevulinate synthetase activator of fresh extracts of semi-anaerobically grown Rhodopseudomonas spheroids was resolved into two fractions by ion-exchange chromatography. One fraction was identified as cystine trisulphide (CySSSCy). Analysis of the other fraction indicated the presence of about equal amounts of glutathione trisulphide (GSSSG) and the mixed trisulphide of glutathione and cystine (GSSSCy). 2. Four further fractions with activator activity were observed on ion-exchange chromatography of extracts prepared by methods similar to those described earlier [Neuberger et al. (1973)Biochem. J. 136,491-499]. These activators were generated by the extraction procedure. Two of them have been identified as trisulphanedisulphonate (S5O62-) and additional cystine trisulphide. 3. For the series of polysulphanedisulphonates (-O3S-Sn-SO3-, n greater than or equal to 1), activator activity at muM concentrations was exhibited only by compounds with n greater than 3. This, together with the results described above, indicates that for a compound R-Sn-R' (where R and R' are organic or inorganic groups) the only structural requirement for activity is n greater than or equal to 3. 4. Oxygenation of a semipanaerobic culture of R. spheroids for 1.5h before harvesting the cells produced a decrease of more than 90% in the cellular content of cystine trisulphide and glutathione trisulphides. 5. Chromatography on DEAE-Sephadex confirmed the presence of multiple forms of aminolaevulinate synthetase in extracts of R. spheroides [Tuboi et al. (1970) Arch. Biochem. Biophys. 138,147-154]. Oxygenation of a semi-anaerobic culture resulted in the disappearance of high-activity enzyme (a-form) and the accumulation of low-activity enzyme (b-form) in the cell. Spontaneous activation [Marriott et al. (1969) Biochem. J. 111,385-394] And activation by cystine trisulphide both resulted in the almost complete conversion of the b-form into the a-form.

Purification and properties of an extracellular xylanase from the thermophilic fungus Humicola grisea var. thermoidea

1991 ◽  
Vol 37 (9) ◽  
pp. 675-681 ◽  
Author(s):  
Rubens Monti ◽  
Héctor Francisco Terenzi ◽  
João Atílio Jorge
Keyword(s):  
Enzyme Purification ◽  
Xylanase Activity ◽  
Sole Source ◽  
Larch Wood ◽  
Purification And Properties ◽  
Sds Page ◽  
Component Form ◽  
Activity Form ◽  
Humicola Grisea ◽  
Temperature Optima

Humicola grisea var. thermoidea mycelium grown on xylan as the sole source of carbon produced at least two extracellular xylanases. The main xylanolytic component (form 2; 90% of recovered activity) was purified to homogeneity. The apparent molecular mass of the purified enzyme was estimated to be 23 000 and 25 550 Da by Bio-Gel P-60 filtration and urea–SDS–PAGE, respectively. The purified enzyme was a glycoprotein with 45% carbohydrate content and pH and temperature optima of 5.5 and 70 °C, respectively. The apparent Km and Vmax values determined with larch-wood xylan were 3.3 mg/mL and 229 μmol∙min−1∙mg protein−1, respectively. The enzyme was highly specific for xylan and degraded this substrate to produce xylo-oligosaccharides, suggesting that it is a β-1,4-endoxylanase (EC 3.2.1.8). The minor enzymatic component of H. grisea extracellular xylanase activity (form 1) was partially purified and some of its properties were studied for comparative purposes. The results obtained suggested that the mode of action of xylanases form 1 and 2 on xylan differs. Key words: xylanase, hemicellulase, enzyme purification, endoxylanase, Humicola grisea.

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