Biomedical subjects
Publications and source records attributed to R N Pau.
For 50 years molybdenum had been considered to have an indispensable catalytic function for nitrogen fixation. Two nitrogenases recently isolated from the bacterium Azotobacter have changed this view. One is a vanadium-containing enzyme and the other lacks both molybdenum and vanadium. Similar nitrogenases may occur in other nitrogen-fixing organisms.
We have constructed a strain of Azotobacter vinelandii which has deletions in the genes for both the molybdenum (Mo) and vanadium (V) nitrogenases. This strain fixed nitrogen in medium that did not contain Mo or V. Growth and nitrogenase activity were inhibited by Mo and V. In highly purified medium, growth was limited by iron. Addition of other metals (Co, Cr, Cu, Mn, Ni, Re, Ti, W, and Zn) did not stimulate growth. Like the V-nitrogenase, the nitrogenase synthesized by the double deletion strain reduced acetylene to both ethylene and ethane (C2H6/C2H4 ratio, 0.046). There was an approximately 10-fold increase in ethane production when Mo was added to the deletion strain grown in medium lacking Mo and V. This change in reactivity may be due to the incorporation of an Mo-containing cofactor into the nitrogenase synthesized by the double-deletion strain. A strain synthesizing the V-nitrogenase did not show a similar increase in ethane production. The growth characteristics of the double-deletion strain, together with the metal composition reported for a nitrogenase isolated from a tungstate-tolerant strain lacking genes for the molydenum enzyme grown in the absence of Mo and V (J. R. Chisnell, R. Premakumar, and P. E. Bishop, J. Bacteriol. 170:27-33, 1988) show that A. vinelandii can synthesize a nitrogenase which lacks both Mo and V. Reduction of dinitrogen by nitrogenase can therefore occur at a center lacking both these metals.
1. Two pro-(phenol oxidase) were distinguished when the blood of late-third-instar larvae of Calliphora erythrocephala was electrophoresed in polyacrylamide gels with Tris-glycine buffer, pH 8.3. One pro-(phenol oxidase), after activation by an enzyme readily catalyses the oxidation of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-dopa). The second enzyme catalyses the oxidation of L-dopa but not of L-tyrosoine. 2. One of the pro-(phenol oxidases) was purified over 2000-fold from homogenates of whole larvae. This enzyme, after activation, catalyses the oxidation of both dopa and tyrosine. On electrophoresis in polyacrylamide gels with Tris-glycine buffer, pH 8.3, it has the same mobility as the enzyme in the blood which catalyses the oxidation of both tyrosine and dopa. 3. The pro-(phenol oxidase)-activating enzyme was purified over 100-fold from homogenates of whole larvae. 4. The oxidation of L-tyrosine, in the presence of the activated purified phenol oxidase, reached a steady maximum rate after a lag period that was directly related to tyrosine concentration and inversely related to enzyme concentration. 5. The effect of the addition of electron donors on the lag period was studied. Dopa, dopamine (3,4-dihydroxyphenethylamine) and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine are the most effective hydrogen donors. 3,4-Dihydroxybenzoic acid, the oxidation of which was not catalysed by the activated pro-(phenol oxidase), did not affect the lag period.
1. The isolation of an o-diphenol oxidase from an acetone-dried powder of late-third-instar larvae of Calliphora erythrocephala was investigated. An insoluble and micro-crystalline fraction containing the enzyme activity was obtained after fractionating extracts of the acetone-dried powder with (NH4)2SO4 and acetone. 2. This fraction can be solubilized in 0.1% sodium dodecyl sulphate without loss of activity. 3. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate shows that the o-diphenol oxidase is a minor component of the extracts from the acetone-dried powder. 4. The o-diphenol oxidase was purified by zonal centrifugation on a sucrose density gradient in the presence of sodium dodecyl sulphate. 5. The amino acid composition of the purified enzyme resembles that of some other o-diphenol oxidases. 6. The subunit composition of the o-diphenol oxidase is discussed.
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