Phosphoenolpyruvate Carboxylase from the Crassulacean Plant Bryophyllum fedtschenkoi Hametet Perrier

ABSTRACrThe carbon isotope fractionation associated with nocturnal malic acid synthesis in Kalanchoe daigremontiana and Bryophyllum tubiflorum was calculated from the isotopic composition of carbon-4 of malic acid, after appropriate corrections. In the lowest temperature treatment (17°C nights, 23°C days), the isotope fractionation for both plants is -4%0(that is, malate is enriched in 13C relative to the atmosphere). For In the case of K. daigremontiana, the isotope fractionation associated with malate synthesis is independent of CO2 concentration. It appears that stomatal aperture decreases as CO2 concentration increases, and the ratio ofexternal CO2 concentration to internal CO2 concentration stays constant, as does the partitioning of the internal CO2 pool (9). Results obtained from isotopic studies have been correlated with results ofgas-exchange studies (7,20).It has been known for some time that nocturnal CO2 fixation contributes decreasingly to net CO2 fixation in CAM plants at higher temperatures (14,15,25). The purpose of this work is to learn about the temperature dependences of individual steps in the CAM pathway. Because our method focuses specifically on malate synthesis, we can learn about how rates of diffusion and carboxylation change as the temperature increases. We also present data concerning changes in the isotopic composition of malate during deacidification which tell about the mechanics of the deacidification process. MATERIALS AND METHODSPlants ofKalanchoe daigremontiana and Bryophyllum tubiflorum were grown from leaflets in a well-ventilated chamber in the University of Wisconsin Biotron with a 10-h light period and a 14-h dark period. The air in the Biotron circulates freely, with a complete turnover in 4 min; Its 6'3C value is -8%o. The plants were watered on alternate days with water and with half-strength Hoagland solution. Three temperature regimes were investigated: the first set of experiments was conducted with 5-month-old plants with a 17°C night temperature and a 23°C day temperature. After sample collection was complete, the temperature was changed to 22°C/28°C and the plants were allowed to adapt for 10 d before samples were taken. After samples were taken at the second temperature, the temperature was changed to 27°C/33°C and the plants were allowed to adapt for 10 d before samples were taken.

Section: Discussionmentioning

confidence: 91%

Temperature Dependence of Carbon Isotope Fractionation in CAM Plants

Deléens

Treichel²,

O’Leary³

1985

“…Malate has long been postulated as a means of regulating the activity PEPc (10), and studies with crude extracts of CAM plant leaves have shown (11,19,22) that during the day the enzyme is strongly inhibited by malate, while after short periods of darkness it is relatively insensitive to inhibition by malate. When PEPc is purified, the sensitivity to malate is rapidly lost (22), although Jones et al (6) found that PEG and desalting ofextracts protected the enzyme from loss of malate sensitivity.…”

mentioning

confidence: 99%

Diurnal Regulation of Phosphoenolpyruvate Carboxylase from Crassula

Wu¹,

Wedding²

1985

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values. Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K,,, for phosphoenolpyruvate and an increased V.,,,,, but the night, or insensitive, form shows only an increase in V,,., in response to glucose-6-phosphate.The daily cycle of nonphotosynthetic CO2 fixation followed by decarboxylation to produce CO2 within leaves closed to gas ' Such a system depends on the sequential use of the product of one set of reactions as the substrate of the next. For example, pyruvate produced by the malic enzyme can be converted to PEP by puruvate kinase to become the substrate of PEP carboxylase. The malate produced by PEPc and malate dehydrogenase from this PEP is the substrate of malic enzyme. Such a sequence of reactions is certain to become a futile cycle unless the components of the system are under rigid control. The central role of malate in such a system is apparent. It has been shown that high concentrations of malate such as those which accumulate during the night in most CAM plants are capable, particularly in conjunction with a low pH, ofconverting the NAD malic enzyme from a low activity dimeric form to a high activity tetramer (21). Malate has long been postulated as a means of regulating the activity PEPc (10), and studies with crude extracts of CAM plant leaves have shown (11,19,22) that during the day the enzyme is strongly inhibited by malate, while after short periods of darkness it is relatively insensitive to inhibition...

“…Reports of interactions of malate with G-6-P are manifold (2,4,5,9,11,12), although they include few examples of studies with even partially purified enzymes. Indeed, a major problem in understanding PEPC regulation has been the transient nature of the characteristics attributable to PEPC and the differing characteristics reported for the enzyme from different species (2,6,7,12). The trend with the CAM enzyme appears to be loss during purification or storage ofthe sensitivity to malate associated with the day enzyme.…”

mentioning

confidence: 99%

Malate Inhibition of Phosphoenolpyruvate Carboxylase from Crassula

Wedding

Black²

1986

ABSTRACIPhosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate2-concentration. Of the reaction components only HC03-increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K.,, but no effect on V,,,,. Enzyme preincubated with malate shows the same effect of malate on K,, but in addition V.. is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows Vtype inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate.