Date of Award:

5-1973

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Plants, Soils, and Climate

Department name when degree awarded

Plant Nutrition and Biochemistry

Committee Chair(s)

Gene W. Miller

Committee

Gene W. Miller

Committee

D. K. Salunkhe

Committee

George W. Chochran

Committee

David R. Walker

Committee

Thomas M. Farley

Abstract

Experiments on the incorporation of 14C-protoporphyrin IX into chlorophyll indicate that ferrous iron may be involved in reactions between protoporphyrin IX and chlorophyll. Experiments using both health and chlorotic tobacco tissue for incorporation studies show that incorporation of protoporphyrin IX into chlorophyll was activated by Fe2+.

S-adenosylmethionine-magnesium protoporphyrin IX methyltransferase (E.C.2.1.1.11) which catalyzes the reaction between S-adenosylmethionine and Mg protoporphyrin IX to form S-adenosylhomocysteine and Mg protoporphyrin IX monomethyl ester was demonstrated in barley seedlings. Methyltransferase was found principally in whole chloroplasts and fractions containing mitochondria were disintegrated chloroplasts. Use of detergent Tween 80, precipitation with protamine sulfate and ammonium sulfate gave a 20-24 fold purification. The purified enzyme exhibited only one protein band after polyacrylamide gel electrophoresis (but the absence of other constituents cannot be concluded).

Both Mg protoporphyrin IX and protoporphyrin IX serve as substrates for the methyltransferase reaction; however, the reaction rate with the latter was only about one-fourteenth of that found with the former. Enzyme activity was proportional to incubation time up to one hour; the enzyme remained active up to 3 hours but decreased sharply in activity with further incubation.

Kinetic studies indicated that methyltransferase is active over a broad pH range with maximum activity around pH 7.5 with Tris-HCl buffer (the enzyme is also active in phosphate buffer at pH 7.5). At pH 7.5 in Tris-HCl buffer and at 37° the apparent Michaelis constant (Km) for Mg protoporphyrin IX and S-adenosylmethionine was 48 and 39 μM, respectively. Only trace enzyme activity remained after being heated at 60° for 5 minutes.

Either Spadenosylethionine or S-adenosylhomocysteine was found to be a competitive inhibitor with respect to S-adenosylmethionine in the methyltransferase reaction, while heroin (ferric protoporphyrin IX) was a non-competitive inhibitor with respect to Mg protoporphyrin IX. Thiol groups were suggested to be essential for the methyltransferase reaction in experiments involving thiol activators and inhibitors.

The enzyme tolerated at a final concentration of 10 per cent methanol, but not acetone or ethanol. Coproporphyrin III (0.10 mM) enhanced enzyme activity whereas protoporphyrin IX (0.20mM) had little effect (although it alone served as a substrate). Chelating agents like diethylenetriaminepentaacetic acid, 1,10-phenanthroline (3 mM) and chromotropic acid (3 mM), and salts such as Fe2+, Fe3+ and F- (each at a concentration of 1 mM) inhibited enzyme activity.

Since the methylation of protoporphyrin IX was inhibited by EDTA and α, α'-dipyridyl (3 mM), this may indicate that metals may be inserted into the protoporphyrin ring before methylation takes place. Aerobic conditions favored the enzyme reaction when protoporphyrin IX or Mg protoporphyrin IX served as substrate.

Four different plant species were tested for methyltransferase activity. The highest enzyme activity was found in cucumber, followed by barley, pea and soybean seedlings.

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