Abstract
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.
Highlights
I. 0-Aminolaevulinate synthetase has been detected in extracts of soybean callus tissues and the enzyme activit\' reached its maximum when callus were I I days old
] y 9, This paper presents the results of an investigation of &aminolaevulinate synthetase activity in extracts of soybean callus cultures which is a vegetable tissue with an active cell division but which fails to synthetize chlorophyll in amounts equivalent to that found in mature leaves
Assay of enzymes 6-Aminolaevulinate synthetase was assayed as follows : 2 ml of reaction mixture containing IOO/~moles of glycine, Ioo/~moles of succinate, Io#moles of ATP, 3.5 nmoles of CoA, io/,moles of MgC12, 0.25/~mole of pyridoxal phosphate, Io #moles of GSH, 5o#moles of Tris-HC1 buffer, I m g of succinyt-CoA synthetase and appropriate amount of extracts, were incubated in a test tube for 30 min at 37 °, the reaction was stopped by addition of 0.5 ml of 25o/o/ trichloroacetic acid. 3-Aminolaevulinate formed was determined according to the method of URATAAND GRANICK12 and identified by thinlayer chromatography:3 of the reaction product itself and of the pyrrole produced from 6-aminolaevnlinate with acetyl-acetone
Summary
Source material of enzyme Callus from soybean seeds was obtained and grown according to MILLER1°. The results obtained were the same whether cells were homogenized or disrupted by ultrasonic treatment, In some cases, extracts were centrifuged at 4° for 5 min at 500 × g immediately afterwards and the supernatant examined for &aminolaevulinate synthetase activity. Assay of enzymes 6-Aminolaevulinate synthetase was assayed as follows : 2 ml of reaction mixture containing IOO/~moles of glycine, Ioo/~moles of succinate, Io#moles of ATP, 3.5 nmoles of CoA, io/,moles of MgC12, 0.25/~mole of pyridoxal phosphate, Io #moles of GSH, 5o#moles of Tris-HC1 buffer (pH: 7.2), I m g of succinyt-CoA synthetase (specific activity 8-12/~moles of succinyl-CoA/h per mg) and appropriate amount of extracts, were incubated in a test tube for 30 min at 37 °, the reaction was stopped by addition of 0.5 ml of 25o/o/ trichloroacetic acid. Protein concentration was measured by the method of LOWRY et a[. 1'5
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