Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi.

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.     

Abnormal chloroplast structures in a mutant of Chlamydomonas reinhardi.

Atypical, highly organized, chloroplast structures, which occurred as tubules or spirals and appeared to be composed of a double membrane, were found in a mutant of Chlamydomonas reinhardi. These structures were light-induced, but their appearance was not directly related to the presence of chlorophyll.     

Extracellular phosphatases of Chlamydomonas reinhardi and their regulation.

Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication.     

Topography of Chlamydomonas: fine structure and polypeptide components of the gametic flagellar membrane surface and the cell wall

Surface polypeptide components of the flagellar membrane of Chlamydomonas reinhardi Dang. gametes are identified by their accessibility to in-vivo vectoral labeling by glucose oxidase-coupled lactoperoxidase-dependent ¹²⁵I iodination. Vectoral labeling is accomplished without observable adverse effects on cell viability or gametic function. Flagella isolated from labeled wild-type cells carry about 3% of the total incorporated label, which is found by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be distributed among 16 identifiable polypeptide bands. The most prominent surface-labeled species migrates in the M_r (relative molecular weight) 350 k region of the gel; each of the remaining iodinated polypeptides, which range in M_r from 25 k to 500 k, carries only a small proportion of incorporated label. To determine which polypeptides are unique to the flagellum and which are contaminants from the cell wall, wild-type profiles were compared with those of mutant strains and of mechanically isolated cell walls. Identification of contaminants was also facilitated by two-dimensional peptide mapping. We conclude that only 11 of the labeled bands are contributed by flagellar polypeptides; the remaining five bands are shown to be contaminants from the cell wall, and additional cell-wall polypeptides are found to co-migrate with flagellar species. A polypeptide designated as a possible membrane tubulin in preliminary studies is shown here to be different from tubulin in its peptide map. The 11 polypeptides assigned as specific flagellar surface components are candidate participants in such biological events as sexual adhesion, flagellar surface motility, and sensory signalling.     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. VII. Photosynthetic Phosphorylation by a Mutant Strain of Chlamydomonas reinhardi Deficient in Active P700

Electron transport activity and absorbance changes associated with P700 were investigated in a mutant strain of Chlamydomonas reinhardi with impaired photosynthesis. This mutant strain, ac-8oa, cannot reduce NADP with electrons from either water or dye and ascorbate, but it has considerable Hill activity. The mutant strain shows none of the absorbance changes characteristic of P700. Although unable to carry out cyclic photosynthetic phosphorylation, ac-8oa is able to synthesize ATP when ferricyanide is provided as an electron acceptor.

These observations lead to the conclusion that a site for the coupling of photosynthetic phosphorylation with electron transport must exist between the 2 photochemical systems.

     

Synthesis of ppGpp and chloroplast ribosomal RNA in Chlamydomonas reinhardi

A material identified as guanosine 5',3'-bis-diphosphate (ppGpp) has been detected in extracts of Chlamydomonas reinhardi ac-20 cells grown under mixotrophic conditions or in arg-2 cells deprived of arginine. The material was acid and base labile, susceptible to alkaline phosphatase, resistant to periodate oxidation, had spectral characteristics of a guanine derivative and comigrated on chromatograms with ppGpp from Escherichia coli. In ac-20 ppGpp may be involved in the control of chloroplast ribosomal RNA synthesis. When ac-20 cells were shifted from mixotrophic to autotrophic conditions, the 32Pi labeling of ppGpp, relative to that of GTP, was reduced, while the specific labeling of chloroplast ribosomal RNA was enhanced. Addition of low concentrations of cycloheximide had somewhat similar effects.     

Responses of a Mutant Strain of Chlamydomonas reinhardi to Prolonged Organotrophic Growth

The responses of the wild type strain and of the y-2 mutant strain of Chlamydomonas reinhardi to long term organotrophic growth were studied. It was shown that wild type can be cultured as an organotroph for at least a month with little decrease in chlorophyll content and no loss of viability. On the other hand, the mutant strain y-2 dies during such organotrophic growth, death beginning after 5 to 6 days in the dark. The kinetics of death indicate that the loss of 95% of the chlorophyll precedes death and that revertants to wild type overgrow such a culture. The results suggest that death of y-2 is correlated with the loss of chlorophyll rather than simple metabolic response to organotrophy and that the chloroplast or a chloroplast related factor may perform certain nonphotosynthetic functions in C. reinhardi. The activities of nicotine adenine dinucleotide and nicotine adenine dinucleotide phosphate dependent triose phosphate dehydrogenases were studied during long term organotrophic growth of y-2. It was found that the activities of these enzymes varied in a manner consistent with previous findings under these conditions. The activity of glutamic dehydrogenase was found to vary as a function of chlorophyll content in the mutant strain y-2.     

Partial purification and characterization of the major AP endonuclease from Chlamydomonas reinhardi

The major AP endonuclease from Chlamydomonas reinhardi has been partially purified and characterized. The enzyme has a molecular weight of about 38 000 as measured by molecular sieving. There is an absolute requirement for a divalent cation, with magnesium being better than manganese. The activity is stimulated by dithiothreitol and Triton X-100. The activity is sensitive to ionic strength, as 50 mM NaCl or KCl results in 70% inhibition. The enzyme is specific for apurinic and apyrimidinic (AP) sites and does not cleave DNA that has been damaged by ultraviolet light, methyl methanesulfonate, osmium tetroxide or sodium bisulfite. There is no deficiency in the AP endonuclease activity in extracts prepared from two mutants of Chlamydomonas that are sensitive to both ultraviolet light and methyl methanesulfonate. There was no evidence for induction of AP endonuclease after exposure of the cells to methyl methanesulfonate.     

Agglutination and glycosyltransferase activity of isolated gametic flagella from Chlamydomonas reinhardii

Isolated flagella from gametes of both mating types (mt⁺ and mt^-) of Chlamydomonas reinhardii were suspended in buffer containing 7% sucrose. After mixing instantaneous agglutination occurred, giving rise to clumps which seem to be stable for at least 24 h. Control experiments show that no aggregates are formed when gametic flagella of one mating type are mixed with flagella prepared from vegetative cells of the other mating type.This in vitro agglutination is inhibited by a number of salt solutions in the same concentration range in which the agglutination of live gametes is affected. Moreover the clumps of flagella tend to disaggregate completely when the salt solutions are added after agglutination has occurred, or by treatment with trypsin. These observations suggest that the in vitro agglutination of isolated gametic flagella indeed reflects their physiological role in the recognition step of the mating process, which appears to be possible without participation of live gametes.We have also investigated the activity of glycosyl transferases on isolated gametic flagella before and during the in vitro agglutination reaction. As there was no detectable increase in the activity of glycosyl transferases, our results do not favour the hypothesis that these enzymes are involved in the primary step of recognition between gametic flagella.Dedicated to Prof. Dr. Otto Kandler on the occasion of his 60th birthday     

Interactions of pseudomonas aeruginosa hemagglutinins with Euglena gracilis, Chlamydomonas reinhardi, and Tetrahymena pyriformis

The galactosephilic and mannosephilic hemagglutinins of Pseudomonas aeruginosa adsorbed onto Euglena gracilis, Chlamydomonas reinhardi, and Tetrahymena pyriformis. Furthermore, peroxidase binding to the 3 protozoan species was shown to be mediated by these lectins. Binding of Pseudomonas lectins to E. gracilis and C. reinhardi caused their specific agglutination, whereas no agglutination was observed with T. pyriformis, even after treatment by papain or by NaF. Added to the culture medium, the Pseudomonas hemagglutins stimulated growth of E. gracilis and T. pyriformis due to their binding to these protozoa; this effect was partly inhibited by the specific sugar.     

Studies on the expression of a mutant with abnormal cell division in Chlamydomonas reinhardi

A mutant of Chlamydomonas reinhardi, in which cell and nuclear division are no longer synchronised, has been compared with wild type with the aim of clarifying the nature of the difference between the two strains. On entry into stationary phase, wild type cultures show a marked increase in protein, RNA and chlorophyll per cell, whereas mutant cultures do not show a comparable increase. The effect of chemicals which may interfere with particular aspects of the cell division process on the expression of the mutant have been studied. Vitamin B12 and the related compounds, benzimidazole, 5,6,dimethylbenzimidazole and cobaltous chloride increase the asynchrony between cell and nuclear division and consequently lead to the accumulation of large multinucleate cells. The mutant is less resistant than wild type to the inhibitory effects of caffeine.