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Limited proteolysis of NADP-malate dehydrogenase from pea chloroplast by aminopeptidase K yields monomers. Evidence of proteolytic degradation of NADP-malate dehydrogenase during purification from pea.

NADP-malate dehydrogenase (L-malate: NADP oxidoreductase, EC 1.1.1.82) from leaves of Pisum sativum has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis. In the crude leaf extract and in the absence of protease inhibitors in the isolation medium, the N-terminus of NADP-MDH was found to be highly susceptible to proteolysis. Evidence of proteolysis during purification includes observations of reduced subunit size on SDS-PAGE and reduced specific activity. Experiments were carried out to investigate the function of the N-terminal amino acid sequence extension of NADP-MDH. Limited proteolysis of highly active (600 units/mg protein) NADP-MDH using aminopeptidase K yielded catalytically active monomers of 34.7 kDa. The results support the conclusions that the N-terminal region is located at the surface of the protein, and that for maintenance of the native NADP-MDH dimer an N-terminal amino acid sequence is important.

Amino Acid Sequence↗

Isoxazolin-5-ones and Amino Acids in Root Exudates of Pea and Sweet Pea Seedlings.

Seeds of Pisum sativum L. cv Finale and Lathyrus odoratus L. cv Spencer were germinated aseptically in moistened sand in the dark. At several stages, the amino acid composition of the exudate and of the corresponding roots was analyzed. A number of common amino acids, including homoserine, were exuded by the growing seedling root in an early stage and were partly reabsorbed later. A number of uncommon amino acids, including several isoxazolin-5-one derivatives, uracil alanines, l-gamma-glutamyl-d-alanine, and alpha-aminoadipic acid were exuded at different rates.

Journal Article↗

Comparative indole-3-acetic Acid levels in the slender pea and other pea phenotypes.

Free indole-3-acetic acid levels were measured by gas chromatography-mass spectrometry in three ultra-tall ;slender' Pisum sativum L. lines differing in gibberellin content. Measurements were made for apices and stem elongation zones of light-grown plants and values were compared with wild-type, dwarf, and nana phenotypes in which internode length is genetically regulated, purportedly via the gibberellin level. Indole-3-acetic acid levels of growing stems paralleled growth rates in all lines, and were high in all three slender genotypes. Growth was inhibited by p-chlorophenoxyisobutyric acid, demonstrating the requirement of auxin activity for stem elongation, and also by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. It is concluded that the slender phenotype may arise from constant activation of a gibberellin receptor or transduction chain event leading directly or indirectly to elevated levels of indole-3-acetic acid, and that increased indole-3-acetic acid levels are a significant factor in the promotion of stem elongation.

Journal Article↗

Effect of field pea replacement level on intake and digestion in beef steers fed by-product-based medium-concentrate diets.

Four ruminally and duodenally cannulated steers (703.4 +/- 41 kg initial BW) were used in a 4 x 4 Latin square to evaluate the effects of field pea inclusion level on intake and site of digestion in beef steers fed medium-concentrate diets. Steers were offered feed ad libitum at 0700 and 1900 daily and were allowed free access to water. Diets consisted of 45% grass hay and 55% by-products based concentrate mixture and were formulated to contain a minimum of 12% CP (DM basis). Treatments consisted of (DM basis) 1) control, no pea; 2) 15% pea; 3) 30% pea; and 4) 45% pea in the total diet, with pea replacing wheat middlings, soybean hulls, and barley malt sprouts in the concentrate mixture. Experimental periods consisted of a 9-d dietary adjustment period followed by a 5-d collection period. Grass hay was incubated in situ, beginning on d 10, for 0, 2, 5, 9, 14, 24, 36, 72, and 98 h; and field pea and soybean hulls for 0, 2, 5, 9, 14, 24, 36, 48, and 72 h. Total DMI (15.0, 13.5, 14.1, 13.5 +/- 0.65 kg/d) and OM intake (13.4, 12.0, 12.6, 12.0 +/- 0.58 kg/d) decreased linearly (P = 0.10) with field pea inclusion. Apparent ruminal (17.5, 12.0, 0.6, 6.5 +/- 4.31%) and true ruminal CP digestibility (53.5, 48.7, 37.8, 46.2 +/- 3.83) decreased linearly (P < 0.10) with increasing field pea. Neutral detergent fiber intake (8.9, 7.9, 7.8, 7.0 +/- 0.3 kg/d) and fecal NDF output (3.1, 2.9, 2.6, 2.3 +/- 0.2 k/d) decreased linearly (P < 0.03) with increasing field pea. No effects were observed for microbial efficiency or total-tract digestibility of OM, CP, NDF, and ADF (P > or = 0.16). In situ DM and NDF disappearance rates of grass hay and soybean hulls decreased linearly (P < 0.05) with increasing field pea. Field pea in situ DM disappearance rate responded quadratically (P < 0.01; 5.9, 8.4, 5.5, and 4.9 +/- 0.52%/h, for 0, 15, 30, and 45% field pea level, respectively). Rate of in situ CP disappearance of grass hay decreased linearly (P < 0.01) with increasing field pea level. Field pea is a suitable ingredient for beef cattle consuming medium-concentrate diets, and the inclusion of up to 45% pea in by-products-based medium-concentrate growing diets decreased DMI, increased dietary UIP, and did not alter OM, NDF, or ADF digestibility.

Animal Feed↗

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants.

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and 1H NMR spectroscopy.

Amino Acid Sequence↗