Changes in the heterogeneity of ribulosebisphosphate carboxylase–oxygenase in winter rye induced by cold hardening

1982 ◽  
Vol 60 (9) ◽  
pp. 897-903 ◽  
Author(s):  
N. P. A. Huner ◽  
D. B. Hayden
Keyword(s):  
Large Subunit ◽  
Cold Hardening ◽  
Winter Rye ◽  
Charge Heterogeneity ◽  
Two Dimensional Gel Electrophoresis ◽  
Bisphosphate Carboxylase ◽  
Ribulose 1 ◽  
Ribulosebisphosphate Carboxylase ◽  
Quaternary Structures ◽  
Molecular Weight Heterogeneity

The quaternary structures of ribulose-1, 5-bisphosphate carboxylase–oxygenase from cold-hardened and unhardened Puma rye were examined by two-dimensional gel electrophoresis according to the method of O'Farrell. The results indicate that major changes in charge heterogeneity occur in the large subunit of this enzyme during growth at cold-hardening temperatures. The extent of charge heterogeneity decreased upon adaptation of Puma rye to cold-hardening temperatures. In addition to charge heterogeneity, molecular weight heterogeneity was also evident in the large subunit polypeptides of the enzyme from cold-hardened and unhardened Puma rye.

Limitation of the Rate of Ribulosebisphosphate Carboxylase Activation by Carbamylation and Ribulosebisphosphate Carboxylase Activase Activity: Development and Tests of a Mechanistic Model

1996 ◽  
Vol 23 (2) ◽  
pp. 141 ◽  
Author(s):  
IE Woodrow ◽  
ME Kelly ◽  
KA Mott
Keyword(s):  
Mechanistic Model ◽  
Large Subunit ◽  
Intercellular Co2 Concentration ◽  
Rubisco Activase ◽  
Photon Flux ◽  
Photon Flux Density ◽  
Bisphosphate Carboxylase ◽  
Ribulosebisphosphate Carboxylase ◽  
Kinetics Of ◽  
Rubisco Activation

A mechanistically-based model of light-mediated activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is developed. The model describes the kinetics of Rubisco activation following a relatively rapid increase in photon flux density (PPFD) from an initially low level. Underlying the model is the assumption that there are two slow processes that could potentially limit the rate of light-mediated Rubisco activation. These processes are the addition of the activator CO2 to the large subunit of Rubisco, which is accompanied by a conformational change in the enzyme (carbamylation), and activase-mediated removal of ribulose 1,5-bisphosphate from the inactive form of the enzyme. The contribution of these slow processes to the overall activation kinetics of Rubisco was resolved by measuring Rubisco activation in whole spinach leaves using non-steady-state CO2 exchange. It was found that when the change in PPFD was relatively small and a correspondingly small proportion of the Rubisco pool was activated, the kinetics of activation were highly sensitive to the intercellular CO2 concentration (ci). The apparent rate constant for activation under these conditions was found to be similar to that for the carbamylation of purified spinach Rubisco. When the change in PPFD and the proportion of Rubisco activated was relatively large, however, the kinetics of Rubisco activation were almost completely CO2 insensitive and were consistent with those of an enzyme-catalysed reaction. It is suggested that (1) CO2-insensitive activation reflects the operation of Rubisco activase and (2) the increasing CO2 sensitivity seen as the change in PPFD decreases reflects a transition to limitation by carbamylation.

Changes in the net charge and subunit properties of ribulose bisphosphate carboxylase–oxygenase during cold hardening of Puma rye

1979 ◽  
Vol 57 (2) ◽  
pp. 155-164 ◽  
Author(s):  
N. P. A. Huner ◽  
F. D. H. Macdowall
Keyword(s):  
Molecular Weight ◽  
Large Subunit ◽  
Sodium Dodecyl ◽  
Reducing Agent ◽  
Cold Hardening ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Bisphosphate Carboxylase ◽  
Thaw Cycle ◽  
Ribulose Bisphosphate Carboxylase Oxygenase

Ribulose bisphosphate carboxylase–oxygenase (RUBPCase) from leaves of cold-hardened and unhardened Puma rye was purified by gel filtration and ion exchange chromatography. The specific activity of the hardened form was twice that of the unhardened form. A difference in charge between the two forms of this enzyme was proved by gel electrofocussing. The estimated isoelectric point (pI) values were 6.4 and 6.3 for the enzyme from the hardened and unhardened source respectively. The large subunit (55 000 molecular weight) of the enzyme from only the unhardened source formed an apparent dimer during sodium dodecyl sulfate (SDS) gel electrophoresis. At pH 6.8 it was also the source of an anomalous polypeptide with an apparent molecular weight of 47 000. This anomalous polypeptide appeared in both hardened and unhardened preparations after irreversible inactivation of RUBPCase activity by NaCl. It also appeared after preparation of the purified enzymes for SDS–PAGE in the absence of β-mercaptoethanol, but this was reversible. The enzyme from the hardened source was less affected in the absence of reducing agent. Structural evidence was obtained for the previously reported cold hardening of the enzyme against freeze inactivation. A freeze–thaw cycle applied to the enzyme in vitro caused some polymerization of the large subunit and its anomalous polypeptide, in the absence of reducing agent, especially in the unhardened case. This increased with repeated cycles until the fifth cycle when the large subunit monomer and its satellite were abolished only in preparations from the unhardened source. These data indicate that the large subunit is a probable site of change that occurred in this enzyme during cold hardening.

Structural Properties and Ribulosebisphosphate Carboxylase and Oxygenase Activity of Fraction-1 Protein from the Marine Alga Halimeda cylindracea (Chlorophyta)

1976 ◽  
Vol 3 (1) ◽  
pp. 93 ◽  
Author(s):  
T Akazawa ◽  
CB Osmond
Keyword(s):  
Large Subunit ◽  
Homogeneous State ◽  
Plant Type ◽  
Precipitation Line ◽  
Carboxylase Activity ◽  
Bisphosphate Carboxylase ◽  
Subunit A ◽  
Fraction 1 Protein ◽  
Oxygenase Activity ◽  
Ribulosebisphosphate Carboxylase

Ribulosebisphosphate carboxylase/oxygenase activity was detected in Halimeda cylindracea and Chaetomorpha crassa. In H. cylindracea carboxylase activity (72-250 micromoles CO2 fixed per hour per milligram chlorophyll) was sufficient to account for measured photosynthetic rates. The activity of the oxygenase was only 1 % that of the carboxylase but otherwise both enzymes showed properties similar to those of the same enzymes in higher green plants. Fraction-1 protein from H. cylindracea was purified to a homogeneous state as tested by poly- acrylamide gel electrophoresis at pH 8.9. The activity of the ribulose-1,5-bisphosphate carboxylase in the purified preparations was 0.1 micromoles CO2 fixed per minute per milligram protein (pH 7.0). The H. cylindracea fraction-1 protein was shown to comprise two subunits, A and B, with molecular weights 5.4 × 104, and 1.35 x 104, respectively, typical of the plant-type ribulose-1,5-bisphosphate carboxylase. The amino acid composition of the large subunit A was similar to that from spinach and Chlorella enzymes, whereas that of the subunit B was markedly distinguishable from the enzymes of other origins. The close resemblance of the H. cylindricea protein to the plant enzymes was further supported by the formation of a spur in the double immunodiffusion precipitation line, indicating probable existence of sequence-homology of the catalytic larger subunit A, typical of the plant-type enzyme molecules.

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