Eva A.Wider De Xifra

A simple method for measuring erythrocyte porphobilinogenase, and its use in the diagnosis of acute intermittent porphyria

Abstract 1. l. A simple method for measuring erythrocyte porphobilinogenase activity has been devised. 2. 2. Red blood cells are hemolysed by three freeze-thaws, the hemolysate is diluted 15 times and only 0.5 ml of this preparation is used for determining porphobilinogenase. in an incubation system also containing porphobilinogen, NaCl. MgCl 2 . Tris-HCl buffer pH 8.2; which is incubated aerobically, in the dark. at 45°. for 1 hr with shaking. 3. 3. The enzymic activity is quenched by adding TCA. 4. 4. Porphyrinogens are oxidized, protein precipitated centrifuged off and uroporphyrins formed are determined spectrophotometrically in the supernatant. 5. 5. The enzyme is stable up to 1 month if stored at —20°.

δ-aminolaevulinate synthetase in extracts of cultured soybean cells

1. 1. δ-Aminolaevulinate synthetase has been detected in extracts of soybean callus tissues and the enzyme activity reached its maximum when callus were 11 days old. 2. 2. The presence of a compound which seems to control δ-aminolaevulinate synthetase activity was demonstrated. The enzyme was present in the soluble fraction and was very labile. 3. 3. When crude extracts or 500 × g supernatant were stored at 4−6°, the apparent activity of δ-aminolaevulinate synthetase increased by as much as 3–6 times, while the activities of δ-aminolaevulinate dehydratase and succinyl-CoA synthetase did not significantly change during the storage. Activation was dependent on concentrations of cells suspensions during disruption and aging. 4. 4. Gel filtration with Sephadex G-25 of 2000 × g supernatants produced an enzyme fraction 30% more active. An increase in enzyme activity was observed when dark-grown callus were exposed to light. 5. 5. The addition of ATP, gibberellic acid and δ-aminolaevulinate to the culture media diminished activity; iron deficiency also produced an δ-aminolaevulinate synthetase less active.

Studies on the reaction mechanism of soybean callus succinyl CoA synthetase. phosphorylation of the enzyme on immobilized adenosine-triphosphate and enzyme attached to a solid support

dence for reaction 1, the phosphorylated enzyme was prepared by reaction of immobilized succinyl CoA synthetase with ATP, providing a very stable form of the phospho-enzyme. The use of solid supports in biochemistry has received much attention recently and many specific applications have been described [l-2] . This report describes the use of insolubilized ATP and water-insoluble succinyl CoA synthetase as a means of obtaining the phosphoryl enzyme, which is considered to be an obligatory intermediate in the reaction catalyzed by this enzyme, and we also show other useful applications of biospecific adsorbents. The mechanism of succinyl CoA,synthetase (succinate:CoA ligase; ADP, EC 6.2.1.5) has been actively investigated in many laboratories [ 3151. A number of authors [S, 6,9,12,16, 171 have provided evidence for a phosphorylated form of the enzyme. Although there are many uncertainties regarding the over-all mechanism, there is agreement on the participation of the phospho-enzyme in the reaction, which may be formulated as follows:

Acute intermittent porphyria--another approach to therapy.

Abstract 1. 1. Acute Intermittent Porphyria patients in early or acute attacks were treated with folic acid (30 mg/day) administered orally for not longer than 10 days. 2. 2. Both clinical and biochemical improvement immediately followed folic acid administration. 3. 3. This is a simple and short-term therapy, which can be used either for treatment or prophylaxis.

Studies on erythrocyte aminolaevulinate dehydratase I. Its purification and possible therapeutic applications

Abstract 1. 1. A method for purifying human erythrocytes ALA-D, using a mixture of n -butanol and chloroform, which denature hemoglobin, followed by ammonium sulphate fractionation and affinity chromatography yielding a 1600-fold purified enzyme, is described. 2. 2. By oxidation of Sephadex G-25 with NaIO 4 , a polyaldehyde, is obtained which can be covalently bound to the ALA-D; however the immobilized enzyme is inactive, because essential ϵ-amino groups at the active site were involved in the coupling. Similar experiments with another enzyme, Rhodanese, resulted in an active insolubilized preparation. 3. 3. By suspending the carrier-enzyme in buffer, slow solubilization with simultaneous release of protein occurs, indicating that this approach might find important therapeutical applications in the treatment of enzyme deficiencies.

Porphyrin biosynthesis—immobilized enzymes and ligands—X. A novel approach to the study of the relationship between the quaternary structure of aminolaevulinate dehydratase and its activity

Abstract 1. 1. Evidence for dissociation, renaturation, re-association and re-hybridization of bovine liver aminolaevulinate dehydratase attached to Sepharose 4B is reported. 2. 2. When insolubilized enzyme was treated with 3 and 6 M urea, non covalently bound subunits were dissociated and detected in the eluate; these subunits can be re-associated into a soluble functioning enzyme with a specific activity close to that of the original pure soluble dehydratase preparation. 3. 3. After being washed with a renaturing buffer mixture, the matrix-bound subunits recovered a level of enzymatic activity equal to 50 and 20% of that of the immobilized native aminolaevulinate dehydratase. 4. 4. The reversibility of the dissociation process was investigated. Bound-subunits dehydratase can associate with nascent soluble bovine liver aminolaevulinate dehydratase subunits in situ. The product of such treatment, bound-re-associated enzyme, has the same activity as that of the original bound-dehydratase. The matrix-bound-dissociated bovine liver enzyme was also re-hybridized with soluble dehydratase subunits from E. gracilis . 5. 5. The apparent K m and optimum pH of the immobilized subunits were the same as those of the bound-octameric enzyme. 6. 6. A scheme is proposed, explaining the sequence of reactions leading from the bound-octameric dehydratase to the possible different derivatives, formed during the dissociation and re-association experiments.

Porphyrin biosynthesis—immobilized enzymes and ligands. IX. Studies on δ-aminolaevulinate synthetase from cultured soybean cells

Abstract Soybean callus δ-aminolaevulinate synthetase (ALA-S) has been covalently attached to Sepharose 4B. The optimal conditions for binding have been determined. The water-insoluble ALA-S retained 40% of the activity of the original soluble preparation, the coupling yield was also high. Sepharose — ALA-S could be stored at 4°C for periods up to 40 days with only 25% loss of activity and it could be repeatedly used with little alteration of its enzymic activity. pH optima of the free and bound enzyme were the same.

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells.

Abstract Soybean callus succinyl CoA synthetase (succinate : CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose · succinyl CoA synthetase can be stored at 4°C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.

Porphyrin biosynthesis in the soybean callus tissue system—XVIII. Levels of succinyl coa synthetase, cysthationase, rhodanese, aminolevulinate synthetase and aminolevulinate dehydratase in clones of different age

Abstract 1. 1. The activity of Succinyl CoA Synthetase (Suc CoA-S), Cysthationase, Rhodanese, Aminolevulinate Synthetase (ALA-S) and Aminolevulinate Dehydratase (ALA-D) was studied in old (405–407 subcultures) and young (34–36 subcultures) soybean callus clones as a function of the days of growing. 2. 2. Suc CoA-S, ALA-S and ALA-D activities were much lower in old than in young callus, while the activity of Cysthationase and Rhodanese was higher in old callus. 3. 3. ALA-S reached its maximum activity when Rhodanese and Cysthationase their minimum, on the 11th day of growth. It is suggested that the cellular content of a possible thio-compound which would regulate ALA-S activity, is controlled through its degradation by Rhodanese.

Porphyrin biosynthesis in rhodopseudomonas palustris—II. Evidence on the existence of a factor regulating aminolevulinate synthetase activity

Abstract 1. 1. No changes in ALA-S activity were observed when different preparations of R. palustris were stored at 4°C for various periods of time. 2. 2. Mixing supernatants from pigmented and decoloured R. palustris cells, showed that the activity of ALA-S was several times higher than expected, suggesting the presence of an activator. 3. 3. Supernatants from photosynthetically and aerobically grown cells were heated and the effect of the protein-free supernatant was tested on both red and white supernatants. The heated supernatant from aerobic cells increased ALA-S when added to red and white preparations, but the heated red supernatant only activated red supernatant and had no action on the white cells enzyme. 4. 4. By gel filtration on Sephadex G-25 of cell free extracts from R. palustris either aerobically or anaerobically grown, a low molecular weight compound was separated, which added back to the homologeous enzyme enhanced its activity confirming the existence of one or two low-molecular weight and heat-stable factors which would act stimulating ALA-S activity. 5. 5. A scheme is proposed to explain the role of these factors on the control of ALA-S in R. palustris .