Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii

Abstract

We have examed the induction of carbonic anhydrase activity in Chlanydomona reiahardtli and have Identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photonutotrophically on 5% C(0, but its synthesis was induced wader low concentrations of (20 (air levels of (20 The photosynthetic characteristics of the green alga Chlamydomonas reinhardtii are dependent upon the C02 concentration experienced by the alga during growth (1, 2). Cells grown at air levels of C02 (0.03%) can utilize low C02 concentrations much more efficiently than cells grown at high levels of C02 (3-5%). Since the C02 concentration during growth has no effect on either the mechanism of photosynthetic C02 fixation or the Km (C02) of the principal C02 fixing enzyme, RuBP4 carboxylase (2), aikgrown algae must employ another method for increasing the efficiency of C02 utilization. Recent work has demonstrated that the appearance of both CA activity and a mechanism for ' C(02(11, 13, 16). While CA and the C, transport system increase following the transfer of C. reinhardtii from high to low levels of C02 and appear to be required for the maintenance of C02 fixation in an environment deficient in CQ, the exact manner in which these activities coordinate Ci accumulation is not fully known.In this study, we have examined the adaptation of C. rein/ardtii to air levels of C02. The photosynthetic capacity of the organism at low C02 concentrations rapidly increases after a shift from growth on high to low C02 and, S h following the transfer, the alga displays photosynthetic characteristics similar to those observed in air-grown cultures. We have compared the kinetic parameters of C02 fixation during adaptation with the induction of CA and have localized and identified the major species responsible for CA activity using both biochemical and immunological procedures. MATERIALS AND METHODSChlamydomonas reinhardtii 2137 mt+ (obtained from Dr. M. Spalding, Michigan State University) and the cell wall-less mutant, CW-15, were cultured axenically in the minimal medium described by Spalding et al. (13) at 28C and a light intensity of 300 ,#E m-2 s-' (400-700 nm). Cultures were vigorously shaken and bubbled with either 5% C02 in air or with air alone. All experiments were performed with cells in early to midphase exponential growth.For the determination of K112 (C02) and the maximal rate of photosynthesis, P,,,r, algae grown under the appropriate conditions were harvested by centrifugation (4,000g), resuspended in C202-free, 20 mm 3(N-morpholino)propanesulfonic acid buffer (pH 7.2), and the rates of 02 evolution at varying HC03-concentrations were measured at saturating light intensity and 25(C with a Clark type 02 electrode. CA activity in cell pressates, prepared as described below, was determined electrometrically and exrs in Wilbur-Anderson units (WA) (9, 18).For studying the induction and purification of CA we used cultures ofthe C. reinhardtii mutant, CW-1S. This mutant lacks a cell wall,...

Light-Induced Carbonic Anhydrase Expression in Chlamydomonas reinhardtii

“…Results in Figure 2 showed an increase in the accumulation of the 37-kD CA monomer 2 h after transfer to low-CO2 condition. The 37-kD protein is generally a broadband, which on some occasions can be resolved as a doublet signal of approximately 36 to 37 kD as previously reported (3,6). It is noteworthy that in the Western blots presented here, except for the 37-kD band no other immunosignals were observed indicating the specificity of the affinity-purified antibody against CA.…”

“…The periplasmic CA has been shown to be important in the supply to the cells of free CO2 from the dehydration of HCO3- (1,25), and has been identified to be a glycoprotein with a subunit molecular mass estimated at 35 to 37 kD (6,29,32). The induction of CA activity under low-CO2 conditions was shown to be inhibited by the protein translation inhibitor, cycloheximide (22,24,32), suggesting that the enzyme was synthesized de novo when the CO2 concentration was lowered.…”

Isolation and Characterization of a Mutant of Chlamydomonas reinhardtii Deficient in the CO2 Concentrating Mechanism

“…reinhardtii, like many other microalgae, can adapt to low CO2 conditions by inducing a CO2 concentrating mechanism (1)(2)(3). One protein that is important in the acquisition of CO2 is the periplasmic carbonic anhydrase (6,11,31). This protein has a monomer molecular weight of 37 kD (31) and facilitates the diffusion of CO2 into the algal cell.…”

The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii

“…Others such as CAH2 showed a decrease in transcript expression over time (class 7). CAH1 and similar proteins have been found in the periplasm and growth medium under ambient-air CO 2 concentrations (Coleman et al, 1984).…”