Porphyrin biosynthesis in the soybean callus tissue system—XV. The effect of growth conditions

doi:10.1016/0020-711X(75)90078-6

International Journal of Biochemistry

Volume 6, Issue 8, August 1975, Pages 591-606

https://doi.org/10.1016/0020-711X(75)90078-6 Get rights and content

Abstract

1.
1. Effects of various factors on chlorophyll, porphyrin and protein content, growth and on the activities of the enzymes involved in the earlier stages of tetrapyrrole synthesis, in cultured soybean cells, were studied.
2.
2. When dark-grown callus was exposed to light, it was found that the amount of porphyrins formed was not altered, but chlorophyll content as well as Succinyl CoA Synthetase (Suc.CoA-S), Aminolevulic Acid Synthetase (ALA-S), Aminolevulic Acid Dehydratase (ALA-D) and Porphobilinogenase (PBGase) activities increased.
3.
3. Addition of Aminolevulic Acid (ALA) to the medium culture, was found to stimulate porphyrin accumulation and to prevent growth; however chlorophyll content was not significantly modified. ALA-S was inhibited while both ALA-D and PBGase activities were enhanced.
The action of puromycin and mitomycin added along with ALA to the media, was also studied, but neither of these inhibitors modified much the effects produced by ALA.
4.
4. Addition of Porphobilinogen (PBG), showed accumulation of uroporphyrin in the tissue; except inhibition of ALA-S, enzymes activities, protein and chlorophyll content were not modified. Evidence obtained would indicate that callus tissue was not permeable to PBG.
5.
5. Omission of iron from the culture medium, produced porphyrin accumulation and prevented growth. It has been consistently found that, the higher the content of porphyrins, the less the callus growth.
Coproporphyrin was the major component of the porphyrins formed in ALA supplemented or iron deficient media.
ALA-S and ALA-D were reduced under iron deficiency.
6.
6. The addition of ATP to the media, did not affect porphyrin, protein, and chlorophyll synthesis, growth or ALA-D, but Suc.CoA-S, ALA-S and PBGase activities diminished.
7.
7. Gibberelic acid produced a measurable increase of PBGase, while diminished Suc.CoA-S and ALA-S.
8.
8. Succinate increased growth and inhibited ALA-S and ALA-D.
9.
9. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP), added to the medium produced accumulation of porphyrins, consequently, ALA-S was greatly inhibited and growth prevented. PBGase was also diminished.
10.
10. Coproporphyrinogenase and Decarboxylases activities were hardly detected in most experiments, and are limiting.
11.
11. The complex pattern of results obtained is discussed.

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  1. δ-Aminolevulinate-dehydratase (ALA-D) from Rhizobium japonicum and Rhizobium meliloti was isolated and some properties were studied.
- 2.
  2. The enzyme from both strains require DTT to maintain full activity and a concentration of about 8 mM is necessary for its maximum expression. Thiol inactivating compounds and heavy metals ions such as Pb²⁺ and Cd²⁺ inactivate ALA-D.
- 3.
  3. The enzyme exhibits Michaelis-Menten kinetics and has an apparent K_m of 0.095 and 0.1–0.37 mM for Rhizobium japonicum and Rhizobium meliloti respectively.
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  4. For both strains the pH profiles show a well denned maximum at about 7.2–7.6 and a second broad peak or shoulder in the range of pH 9–10.
- 5.
  5. ALA-D from Rhizobium does not appear to be a heat stable enzyme as it happens to be in other sources.
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  1. Properties of porphobilinogenase (PBGase), the enzyme complex converting porphobilinogen (PBG) into uroporphyrinogens, were studied in a wild strain, D273-10B and a mutant, B231, of Saccharomyces cerevisiae
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  2. A well-defined maximum of enzyme activity was observed for the mutant strain after 20 hr of growth; whilst the activity in the wild strain did not vary significantly during growth.
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  3. Neither PBG consumption nor uroporphyrinogen formation were modified by the presence of air either in the wild or in the mutant strain.
- 4.
  4. In both the wild and mutant strains uroporphyrinogen formation increased linearly with both protein concentration and incubation time.
- 5.
  5. The addition of a mixture of sodium and magnesium salts to the assay system inhibited the enzyme activity of both strains by 50% without modifying the isomer composition.
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  6. The same optimum pH (7.4) and mol. wt ( $50,000 ± 5000$ ) was found for the enzyme from both strains.
- 7.
  7. The enzyme from both the wild and mutant strains shows Michaelis-Menten kinetics when isolated from cells at either the exponential or the stationary phases of growth. Accumulation of porphyrins and δ-aminolevulinic acid occurring during the exponential phase in the mutant strain, did not modify the kinetics.
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  1. The effect of light and growing time on chlorophyll, protein content and the activity of porphobilinogenase (PBGase), in Euglena gracilis was studied.
- 2.
  2. It was found that neither protein nor chlorophyll content or PBGase activity were dependent upon the days of growing or the light conditions of growth.
- 3.
  3. Some properties of PBGase were studied. The addition of sodium and magnesium salts to the assay system did not modify enzyme activity. Uroporphyrinogen formation was linear with protein over a wide range of enzyme concentration.
- 4.
  4. When activity was expressed on the basis of the amount of porphyrins formed an optimum pH of 7.4 was obtained but when it was expressed in terms of PBG consumed two pH maxima at 7.0 and 8.5 were observed.
- 5.
  5. Direct plots of velocity against PBG concentration, when activity was measured as PBG consumed, were not hyperbolic; double-reciprocal plots were not linear and Hill plots gave a n value of 2, indicating the existence of positive co-operative effects between two binding sites for PBG per molecule of PBGase.

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Porphyrin biosynthesis in the soybean callus tissue system—XV. The effect of growth conditions

Abstract

Biochem. biophys. Res. Commun.

J. Chromatogr.

J. biol. Chem.

Biochim. biophys. Res. Commun.

J. Chromatogr.

Biochim. biophys. Acta

Archs Biochem. biophys.

Biochim. biophys. Res. Commun.

J. biol. Chem.

FEBS Lett.

FEBS Lett.

Biochim. biophys. Acta

Biochim. biophys. Acta

Phytochemistry

J. biol. Chem.

Biochim. biophys. Acta

Phytochemistry

Phytochemistry

Biochem. biophys. Acta

Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris

PI. Physiol. Lanc.

Study of the separation of uroporphyrin octametyl esters I and III by thin layer and paper chromatography

J. Chromatogr.

Porphyrin biosynthesis I—Studies on erythrocyte preparations

Biochim. biophys. Acta.

Purification and properties of Coproporphyrinogenase

Biochem. J.

Chemistry and Biochemistry of Plant Pigments

L'acide gibbérelique

Endeavour

Studies on S-Adenosylmethionine-Magnesium Protoporphyrin Methyltransferase on Euglena gracilis Strain

Z. Biochem. J.

Paper Chromatography separation of the coproporphyrin isomers I and III

Scand. J. clin. Lab. Invest.

The effects of adenosine triphosphate on Porphyrin excretion and on glycine metabolism in Rh. spheroides

Biochem. J.