Active pyruvate, P1 dikinase in leaf or chloroplast extracts isolated from illuminated leaves was inactivated by incubating with ADP. With chloroplast extracts neither ATP nor AMP alone was effective. Half the maximum rate of inactivation was observed with about 55 μM ADP. The following evidence supported the view that ADP-mediated inactivation had a co-requirement for low concentrations of ATP [Buchanan (1980) Ann. Rev. Plant Physiol. 31: 341], adding hexokinase and glucose prevented inactivation by ADP [Feldhaus et al. (1975) Eur. J. Biochem. 57: 197], when GDP and UDP were added in place of ADP they mediated rapid inactivation only when ATP was also provided; GTP was not effective. ATP was apparently optimally effective at about 1 μM or less. The rate of inactivation was approximately proportional to the square of extract concentration suggesting dependancy on a factor in the extracts in addition to active enzyme. The involvement of one or more heat labile protein factors was confirmed by trypsin treatment of extracts. Pyruvate, P1 dikinase inactivated by treatment with ADP was reactivated by incubating with P1, a property common to the inactive enzyme extracted from darkened leaves. Thiol/disulphide interconversion was apparently not critical in the regulation of pyruvate, P1 dikinase.

An antibody for phosphoenolpyruvate carboxylase was used to isolate and to quantitate the enzyme from greening maize (cv. KOU 6) leaves. The increase in enzyme activity during greening was due to de novo synthesis, which was paralleled by increases in enzyme protein and incorporation of leucine. The light-induced activity was due to one specific isoenzyme. The action spectrum for enzyme synthesis had red and blue peaks.

The cold lability of pyruvate, Pi dikinase in crude leaf extracts was studied in a number of C4 plants. The survey included C4 monocots and dicots and species representingthe three C4 subgroups: NADP-malic enzyme, NAD-malic enzyme, and PEP-carboxykinase types. In some species (e.g., Digitaria sanguinalis, Sorghum bicolor and Echinochloa crus-galli), the enzyme was very sensitive to cold treatment (half life of about 8 min at 0°C and 10 to 15 min at 10°C). In other species (Panicum miliaceum, Panicum maximum and Panicum texanum), the enzyme was very cold tolerant (retention of 60 to 85% activity after 60 min at 0°C and 90% activity after 60 min at 10°C). Among the plants examined, the most cold sensitive pyruvate,Pi dikinase was found among species of the NADP-malic enzyme subgroup.

Cold lability of pyruvate, orthophosphate dikinase was investigated using a homogeneous, purified enzyme preparation from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. Its stability was markedly reduced below about 10 C and the rate of cold inactivation followed first order kinetics at a concentration lower than about 0.1 milligram of enzyme per milliliter. Cold inactivation was little affected by pH in the range which gives good stability for the enzyme at warm temperatures and the enzyme activity was protected strongly by inclusion of substrates (pyruvate and phosphoenolpyruvate) and polyols such as sucrose, sorbitol, and glycerol. Loss of catalytic activity was accompanied by an apparent dissociation of a tetrameric form of the enzyme (9S form) into a new, more slowly sedimenting (5.1S) component. Inclusion of pyruvate at 4 mm in the coldtreated enzyme had no effect on the sedimentation value. A sharp change in activation energy of the dikinase-catalyzed reaction was observed near 12 C and its break point appears to be close to the generally accepted critical low temperature limit for the growth of maize plants.

Journal Article Differing sensitivity of pyruvate orthophosphate dikinase to low temperature in maize cultivars Get access Tatsuo Sugiyama, Tatsuo Sugiyama Department of Agricultural Chemistry, School of Agriculture Shizuoka University836 Ohya, Shizuoka 422, Japan Search for other works by this author on: Oxford Academic Google Scholar Keiko Boku Keiko Boku Department of Agricultural Chemistry, School of Agriculture Shizuoka University836 Ohya, Shizuoka 422, Japan Search for other works by this author on: Oxford Academic Google Scholar Plant and Cell Physiology, Volume 17, Issue 4, August 1976, Pages 851–854, https://doi.org/10.1093/oxfordjournals.pcp.a075342 Published: 01 August 1976 Article history Received: 24 March 1976 Published: 01 August 1976

Phosphoenolpyruvate carboxylase has been purified to homogeneity from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. The ratio of specific activities in crude extracts and the purified enzyme suggests that the enzyme is a major soluble protein in the tissue. The enzyme has a sedimentation coefficient (s(20,w)) of 12.3S and a molecular weight, determined by sedimentation equilibrium, of 400,000 daltons. Dissociation of the enzyme and electrophoresis on dodecyl sulfate polyacrylamide gels yields a single stained band which corresponds to a subunit weight of 99,000 daltons. Thus it appears that the native enzyme is composed of four identical or similar polypeptide chains.The enzyme yields cooperative rate-concentration plots (Hill number of 2) with phosphoenolpyruvate as the variable substrate at pH 7. This cooperativity disappears in the presence of an activator, glucose-6-P, or by raising the pH of the assay mixture to 8. Glycerol (20%, v/v) exerts a similar effect. The enzyme is also activated in the presence of glycine which causes an increase in V(max) without significant effect on the apparent Km for phosphoenolpyruvate and Hill number. The apparent Km for HCO(3) (-) is 0.02 mm, and the activation constant for Mg(2+) is 1.54 mm at pH 7. There is an abrupt discontinuity in Arrhenius plots and an associated increase in activation energy below 10.8 C.