PURIFICATION OF RIBULOSE 1,5‐BISPHOSPHATE CARBOXYLASE AND CARBON ISOTOPE FRACTIONATION BY WHOLE CELLS AND CARBOXYLASE FROM CYLINDROTHECA SP. (BACILLARIOPHYCEAE.)^1,2,3

Abstract

Ribulose 1,5‐biphosphate carboxylase has been purified to homogeneity from extracts of Cylindrotheca sp. (strain N‐1), a marine, pennate diatom. The carboxylase has a molecular weight and structural composition similar to the enzyme from higher plants. When assayed in the presence of 1 mM NaHCO3 the enzyme was stimulated nearly 40% by 1 mM aspartate and over 20% by 1 mM malate, and was inhibited to over 60% by 1 mM phosphoenolpyruvate. Similar experiments, using spinach carboxylase, failed to show activation by these metabolites. When assayed in the presence of 20 mM NaHCO3, 6‐phosphogluconate (1 mM) inhibited activity of ribulose bisphosphate carboxylase from Cylindrotheca by 60%, and higher concentrations of maiate (10 mM) inhibited activity by 25% Carbon isotope fractionation by ribulose bisphosphate carboxylase was ‐32.6% (ppt) when measured under N2 using homogeneous enzyme, whereas maximum carbon isotope fractionation by the whole alga grown in 1% ‐C02‐in air averaged ‐ 16.8%. Carbon isotope fractionation by the whole alga varied with the density of the culture and was maximum at a low cell density (1.7 ± 106 cellslml). At higher densities, the fractionation decreased by 4.0%. Carbon isotope fractionation has been used previously to determine the pathway of carbon metabolism in other organisms; the results of this investigation seem to indicate that this strain uses both the reductive pentose phosphate pathway and the C4 carbon pathway for primary CO2 fixation.

Ribulose Bisphosphate Carboxylase from Three Chlorophyll c-Containing Algae

“…Therefore, the sedimentation coefficients of pea and chromophyte RuBPCase are nearly the same (18S). These results, indicating that RuBPCase holoenzyme from chromophytes has a similar molecular size as the holoenzyme from higher plants (=550 kD), are in agreement with those of Estep et al (7) who found RuBPCase activities from the diatom Cylindrothaca and spinach comigrate during nondenaturing PAGE. Estep et al (7) and Plumley (23) also showed that Cylindrotheca RuBPCase has two classes of subunits, similar in molecular size to the subunits isolated from higher plant RuBPCase.…”

“…chromophytes) has not been well characterized. Studies of one chromophyte, the diatom Cylindrotheca fusiformis, indicate that the RuBPCase holoenzyme has the same mol wt and subunit composition as plant RuBPCase (7,23), but its enzymic activity may be regulated differently than that from higher plants. Metabolites such as malate, aspartate and phosphoenolpyruvate appear to be effectors of the activity of diatom, but not spinach, RuBPCase.…”

Cloning and expression of the chloroplast-encoded rbcL and rbcS genes from the marine diatom Cylindrotheca sp. strain N1

“…strain Nl was shown to be a typical L,S, enzyme [5]. Since diatoms also belong to the Chromophyta, we considered the possibility that the rbcL and rbcS genes might be adjacent and encoded by the ctDNA in strain N 1.…”

13C/12C fractionation by marine diatoms

“…Low CO2 cultures were thosc with 2000 to 4000 pM DIC concentrations. Sources for culture stud~es include: Degens et al (1968a) (excluding air-grown S. costatum), Estep et al (1978), Wong & Sackett (1978), Johnston & Raven (1992, Hinga et al (1994) (excluding experimental run 12), Thompson 8 Calvert (1994), and Laws et a1 (1995). Sources for field studies include: Degens et al (1968b), Deuser (1970), Gearing et al (1984, Hedges et al (1988), Duggins et al (1989), Goering et al (1990), Fischer (1991), Fry & Wainright (1991), Voss (1991), Fogel et al (1992, Ostrom (1992), Nakatsuka et al (1992), Rau et al (1992), Francois et al (1993), Simenstad et al (1993) for Arctic 6I3C-DIC measurements, J.…”