RESUMEN
Mitochondria isolated from etiolated shoots of corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), soybean (Glycine max L. Merr.), and mung bean (Phaseolus aureus) exhibited a proline-dependent O(2) uptake subject to respiratory control. ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on O(2), but not NAD. The major ninhydrin-positive product formed via Delta(1)-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO(2), organic acids, and aspartate were recovered after [(14)C]proline feeding experiments. These results demonstrate the mitochondrial association and O(2) dependence of plant proline metabolism.
RESUMEN
The objective of these experiments was to determine the fate of tritium from the 5 position of proline and to assess the validity of its loss to H(2)O as a measure of proline oxidation. When [5-(3)H]proline was fed to barley (Hordeum vulgare) leaves, tritium was recovered in H(2)O and metabolites such as glutamate, glutamine, organic acids, aspartate, asparagine, and gamma-aminobutyrate. Collectively these metabolites, which are oxidation products of proline, accounted for 8% of the (3)H recovered after 5 hours. In spite of the amount recovered in metabolites, the rates of proline oxidation estimated by measuring (3)H(2)O recovery from [5-(3)H]proline were only slightly lower than rates estimated by incorporation of (14)C into oxidized products and loss of (14)C from total proline. Therefore, (3)H(2)O recovery from [5-(3)H]proline is useful in assessing the effects of stress on proline metabolism.Water stress inhibited proline oxidation, as reported previously. In addition, a reconversion of proline oxidation products to proline occurred in stressed leaves. This observation probably indicates a breakdown in cellular compartmentation of proline synthesis and proline oxidation.
RESUMEN
Barley (Hordeum vulgare cv Prior) leaves converted l-U-(14)C-arginine to labeled proline. Accumulation of radioactivity in proline was greater in wilted leaves, but only after 9 hours of incubation. As the increase in free proline was detectable after only 3 to 6 hours, it is likely that the observed stimulation of proline labeling represents a result rather than a cause of proline accumulation. Furthermore, the loss of total arginine during water stress was only 10 to 15% of the increase in proline. We conclude that arginine probably contributes less than 1% of the carbon in the expanding proline pool of wilted barley leaves.
RESUMEN
Barley (Hordeum vulgare L. var. Prior) leaves converted more (14)C-glutamic acid to free proline when water-stressed than when turgid; neither decreased protein synthesis nor isotope trapping by the enlarged free proline pools found in wilted tissue seemed to account for the result. This apparent stimulation of proline biosynthesis in wilted leaves was not observed when radioactive ornithine or P5C (Delta(1)-pyrroline-5-carboxylate, an intermediate following glutamate in proline synthesis) were used as proline precursors unless proline levels were high as a result of previous water stress. We interpret this to mean that any stimulation of proline synthesis by water stress must act on P5C formation rather than its reduction to proline. Experiments showing greater apparent conversion of (14)C-glutamate to proline do not unequivocally prove that proline synthesis is stimulated by water stress, as P5C feeding studies show that proline oxidation is inhibited under comparable conditions. This inhibition could account, at least in part, for increased proline labeling, and must be considered an alternate possibility.
RESUMEN
Ornithine transcarbamoylase (carbamoyl phosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) has been partially purified from the blue-green alga Nostoc muscorum Kützing, an organism in which the enzyme seems to be involved in a bicarbonate-fixing pathway leading to citrulline. Pertinent to possible regulation of this pathway, the enzyme shows hyperbolic substrate kinetics, has a molecular weight estimated at 75,000 daltons, and its catalytic capability is little influenced by a selection of metabolites that might conceivably act as regulators in vivo. Thus it seems unlikely that this enzyme is the control point for bicarbonate fixation. In terms of energy of activation (12.3 kcal/mole), size and Km for carbamoylphosphate, the Nostoc enzyme resembled preparations from liver and higher plants more than preparations from Streptococcus and Mycoplasma. The enzymes from Streptococcus and Mycoplasma are probably specialized for citrulline breakdown rather than citrulline synthesis. The Km for ornithine was 2.5 mm at a saturating concentration of carbamoylphosphate and the Km for carbamoylphosphate was 0.7 mm at an ornithine concentration of 2 mm. Ornithine was inhibitory at concentrations greater than 2 mm. Phosphate was a competitive inhibitor with respect to carbamoylphosphate. The pH optimum for citrulline synthesis was 9.5.
RESUMEN
The characteristics of the enzyme Delta(1)-pyrroline-5-carboxylic acid dehydrogenase from etiolated barley (Hordeum distichum) shoots have been examined. The bulk of the enzyme activity was found in the 10,000g pellet fraction, this activity being displayed only after detergent treatment of the suspended pellet. The enzyme was most active at pH 8, and activity was NAD-dependent. Enzyme activity was unaffected by either mannitol or sucrose in the reaction mixture up to a concentration of 0.45 m but was strongly inhibited by Cl(-) and, to a lesser extent, SO(4) (2-). The inhibition attributable to KCl was reversed by increasing the concentration of Delta(1)-pyrroline-5-carboxylic acid in the reaction mixture.