Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv 'Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25,23,20,16,12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pl = 8.9) and 60 kilodaltons (anionic, pI = 4.0).The observation that V5S1Na2SO4 was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues.Ethylene is known to accelerate senescence in plants (1). While the loss of RNA, Chl, and protein during senescence is known (17,22), the enzymology of the degradation process is not understood. The original purpose of the work reported here was to characterize the enzymes involved in nucleic acid degradation. As an experimental system, we chose to measure enzyme changes during ethylene-enhanced senescence in cotyledons of 2-weekold cucumber seedlings since Lewington et al. (9) reported that yellowing of cucumber cotyledons was associated with a decrease in RNA, DNA, and protein and with an increase in RNase and DNase activity. While new proteins were detected in SDS-PAGE gels during senescence, none were associated with nuclease activity as measured in SDS-PAGE nuclease activity gels. However, two proteins with red chromaphores, which were subsequently found to be peroxidases, appeared during ethylene-induced senescence. This paper describes the isolation and purification of these enzymes.In spite of the fact that plant peroxidases have been studied by many workers, their physiological functions are only partially understood (7). One function ascribed to peroxidase is that of a Chl-degrading enzyme (12,23 Evidence has accumulated that peroxidase-based reactions may play a role in senescence. Lauriere (8) showed that peroxidase activity often increased during senescence. More specifically, Ford and Simon (6) have reported that peroxidase activity increased during senescence of cucumber plants. Thre...

Infections or inflammatory states often cause significant increases in serum phenylalanine and the phenylalanine-tyrosine ratio. More than 95% of samples obtained during inflammatory diseases in man showed phenylalanine-tyrosine ratio increases greater than the maximum normal values. An increase in this ratio also occurred in monkeys with induced Rocky Mountain spotted fever, viral encephalitis, yellow fever, or pneumococcal and Salmonella infections, as well as in rats with pneumococcal and Salmonella infections, as well as in rats with pneumococcal, Salmonella or tularemia infections. A similar ratio increase occurred in rats inoculated with unpurified mediator substances (released by activated leukocytes) that appear to initiate many of the secondary metabolic phenomena associated with infection and/or inflammation. To identify responsible mechanisms, rats were given lethal doses of Streptococcus pneumoniae; serum phenylalanine and phenylalanine-tyrosine ratios increased significantly. Hepatic phenylalanine hydroxylase activities were slightly decreased when compared to noninfected controls. Infected and noninfected rats showed comparable oxidation rates for 14C-phenylalanine given with an oral phenylalanine load, as a pulse-oral dose, or as an intraperitoneal injection. After 8 hr, both infected and control rats had similar amounts of radioactivity in total body protein, but tissue distributions were markedly altered during pneumococcal sepsis. Serum proteins of infected rats contained almost twice as much total radioactivity as that found in controls, while the amount of labeled phenylalanine in skeletal muscle protein was significantly reduced in the infected group. Isolated muscles from infected rats released more phenylalanine and less tyrosine than control muscles. Infection-related increases in serum phenlalanine could not be explained by decreased hydroxylation or oxidation. Rather, the data were consistent with an increased flux of phenylalanine into serum, most likely as the result of increased skeletal muscle catabolism. Elevations in the serum phenylalanine-tyrosine ratio have potential value for estimating the presence of an inflammatory fisease and the catabolic state of a patient.

Hypozincemia was induced in rats by Salmonella typhimurium and live vaccine strain Francisella tularensis (LVS) infections. Hepatic synthesis of zinc-binding proteins (ZBP) was studied in order to elucidate the mechanisms involved in the redistribution of zinc from plasma to liver occurring during infectious illness. ZBP, labeled in vivo with 65Zn, were isolated and identified as metallothioneins based, in part, on their heat stability, dimorphism, and amino acid composition. Cysteine was the major amino acid found in both forms of metallothionein and constituted 28-31% of total residues. The apparent half-life of these proteins as measured by disappearance of 65Zn was determined to be 19 h in a relatively mild infection (LVS) and 38 h in a more severe S. typhimurium infection. Results provide evidence that metallothioneins not only have the previously postulated regulatory role in normal zinc homeostasis but are intimately involved in the zinc redistribution occurring during the acute stage of infectious illness.

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