Protein variation in the venom spat by the red spitting cobra, Naja pallida (Reptilia: Serpentes).

The venom spat by red spitting cobras (Naja pallida) was analyzed to document variations in protein composition occurring over short temporal periods (less than 5 min). These cobras exhibited distinct control of venom flow with spits averaging 1.7% of the volume of the venom gland, thus enabling the cobras to rapidly expel over 40 consecutive spits. Variations in the low and high molecular weight proteins were observed when comparing the 1st, 20th and 40th spits produced by the same specimens. The first few spits were characterized by a distinctive 9 kDa protein which was never observed beyond the 7th spit, was present in milked venom and was present when the spitting behavior was preceded by a 5 min period of induced defensive behaviors.

A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.

A 29.5 kDa intracellular alpha-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.

Intracellular carbonic anhydrase of Chlamydomonas reinhardtii.

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified to homogeneity from a mutant strain of Chlamydomonas reinhardtii (CW 92) lacking a cell wall. Intact cells were washed to remove periplasmic CA and were lysed and fractionated into soluble and membrane fractions by sedimentation. All of the CA activity sedimented with the membrane fraction and was dissociated by treatment with a buffer containing 200 mM KCI. Solubilized proteins were fractionated by ammonium sulfate precipitation, anionic exchange chromatography, and hydrophobic interaction chromatography. The resulting fraction had a specific activity of 1260 Wilbur-Anderson units/mg protein and was inhibited by acetazolamide (50% inhibition concentration, 12 nM). Final purification was accomplished by the specific absorption of the enzyme to a Centricon-10 microconcentrator filter. A single, 29.5-kD polypeptide was eluted from the filter with sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer, and a 1.5 M ammonium sulfate eluate contained CA activity. In comparison with human CA isoenzyme II, the N-terminal and internal amino acid sequences from the 29.5-kD polypeptide were 40% identical with the N-terminal region and 67% identical with an internal conserved region. Based on this evidence, we postulate that the 29.5-kD polypeptide is an internal CA in C. reinhardtii and that the enzyme is closely related to the alpha-type CAs observed in animal species.

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii with a Carbonic Anhydrase-Directed Photoaffinity Label.

A carbonic anhydrase (CA)-directed photoaffinity reagent, 125I-labeled p-aminomethylbenzenesulfonamide-4-azidosalicylamide,was synthesized and shown to derivatize periplasmic CA in the unicellular green alga Chlamydomonas reinhardtii. The photoderivatization of purified C. reinhardtii periplasmic CA or intact C. reinhardtii cells with the reagent resulted in the modification of the large (37 kD) subunit of the enzyme. Photoderivatization of proteins in lysed C. reinhardtii cells also resulted in the specific labeling of a polypeptide of 30 kD. Centrifugation of the cell extract prior to photoaffinity labeling revealed that the labeled peptide was present predominantly in a particulate fraction. The photoaffinity-labeled 30-kD polypeptide was not observed in extracts from a mutant of C. reinhardtii that is believed to be deficient in an intracellular form of CA. These results provide evidence that the 30-kD polypeptide, which is photoaffinity labeled in lysed C. reinhardtii cells, is an intracellular form of CA.

Effect of dithiothreitol on the catalytic activity, quaternary structure and sulfonamide-binding properties of an extracellular carbonic anhydrase from Chlamydomonas reinhardtii.

Extracellular carbonic anhydrase from the unicellular green alga Chlamydomonas reinhardtii is an oligomeric protein containing subunits of 36 and 4 kDa which are joined by disulfide bonds to form higher molecular mass oligomers. In this study, the effect of dithiothreitol on some properties of the enzyme were examined. Dithiothreitol caused a 40% activation of the catalytic activity of the enzyme at low concentrations (0.1 mM), but an inactivation of about 85% of the catalytic activity at high (50 mM) concentrations. Chemical cross-linking of the enzyme with dimethyl suberimidate revealed the existence of oligomers containing up to three large subunits and at least two small subunits. Cross-linking analysis of dithiothreitol-treated carbonic anhydrase revealed that 0.1 mM dithiothreitol had no effect on the subunit composition of the enzyme, but 10 or 50 mM caused subunit dissociation, including the apparent complete dissociation of the small subunits from the large subunits. There was a characteristic enhancement of dansylamide fluorescence when this fluorescent sulfonamide bound carbonic anhydrase and the fluorescence enhancement was retained following the dithiothreitol-induced dissociation of the enzyme. These results indicate that disulfide bonds are essential for maintenance of the oligomeric structure of Chlamydomonas reinhardtii carbonic anhydrase, and that the small subunit may be necessary for enhancing catalysis, but not for the binding of sulfonamides to the enzyme.

Salt-Induced Dissociation of Carbonic Anhydrase from Intact Cells of Chlamydomonas reinhardtii.

The extracellular carbonic anhydrase of Chlamydomonas reinhardtii is dissociated from either intact or lysed cells by treatment with a 20 millimolar potassium phosphate buffer containing 0.4 molar KCI at pH 7.4. Electrophoretic analysis of proteins dissociated by the high salt treatment reveals that carbonic anhydrase comprises over 70% of the total released. These results suggest that the extracellular carbonic anhydrase in C. reinhardtii is bound to either the cell wall or plasma membrane through ionic interactions.

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme.

A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.

Isolation and Characterization of a Mutant of Chlamydomonas reinhardtii Deficient in the CO(2) Concentrating Mechanism.

A Chlamydomonas reinhardtii mutant has been isolated that cannot grow photoautotrophically on low CO(2) concentrations but can grow on elevated CO(2). In a test cross, the high CO(2)-requirement for growth showed a 2:2 segregation. This mutant, designated CIA-5, had a phenotype similar to previously identified mutants that were defective in some aspect of CO(2) accumulation. Unlike previously isolated mutants, CIA-5 did not have detectable levels of the periplasmic carbonic anhydrase, an inducible protein that participates in the acquisition of CO(2) by C. reinhardtii. CIA-5 also did not accumulate inorganic carbon to levels higher than could be accounted for by diffusion. This mutant strain did not synthesize any of the four polypeptides preferentially made by wild type C. reinhardtii when switched from an environment containing elevated CO(2) levels to an environment low in CO(2). It is concluded that this mutant fails to induce the CO(2) concentrating system and is incapable of adapting to low CO(2) conditions.

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas.

Inorganic carbon (C(i)) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO(2). Both air-grown cells, that have a CO(2) concentrating system, and 5% CO(2)-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C(i)) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO(2)-grown cells also accumulated some C(i), although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO(2) fixation by high CO(2)-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO(2)-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.

Effect of Osmotic Stress on Carbon Metabolism in Chlamydomonas reinhardtii: Accumulation of Glycerol as an Osmoregulatory Solute.

NaCl, KCl, and sucrose at equiosmolar concentrations had similar inhibitory effects on photosynthetic carbon metabolism by the freshwater green alga, Chlamydomonas reinhardtii. Inhibitory concentrations of these solutes altered the products of photosynthetic (14)CO(2) incorporation, resulting in reduced incorporation into starch, sugar phosphates, lactate, and glycolate, but caused an accumulation of glycerol both intracellularly and in the medium.

Muscle cell cultures from chicken breast muscle have increased specific activities of creatine kinase when incubated at 41 degrees C compared with 37 degrees C.

Chicken muscle cell cultures were incubated at 41 degrees C, the physiological chicken body temperature, and compared with cultures incubated at 37 degrees C, the typical cell culture incubation temperature. The cultures incubated at 41 degrees C show not only an increase in creatine kinase (CK)-specific activity but also a marked increase in the percentage of adult muscle CK isozyme (MM-CK) in 7-day muscle cultures. Muscle cell cultures incubated in the presence of cytosine arabinoside (ara-C), a cell proliferation inhibitor, do not have the mononucleated cell overgrowth seen at 41 degrees C and thus exhibit a further increase in creatine kinase-specific activity compared with cultures incubated at 41 degrees C in the absence of ara-C. These results suggest that muscle cell cultures incubated at 41 degrees C are more highly differentiated than those incubated at 37 degrees C.

Properties of Phosphoglycolate Phosphatase from Chlamydomonas reinhardtii and Anacystis nidulans.

The levels of activity of 2-phosphoglycolate phosphatase in the green algae, Chlamydomonas reinhardtii and Chlorella vulgaris, were in the range of 37 to 60 micromoles per milligram chlorophyll per hour and in the blue-green algae, Anacystis nidulans and Anabaena variabilis were 204 to 310 micromoles per milligram chlorophyll per hour. The activity in each species was similar regardless of whether the algae were grown with air or 5% CO(2) in air. The enzyme purified 530-fold from Chlamydomonas was stable, had a broad pH optimum between 6 and 8.5, and was specific for the hydrolysis of P-glycolate with a K(m) of 23 micromolar. The enzyme purified 18-fold from Anacystis was labile, had a sharp pH optimum at 6.3, and was also specific for P-glycolate with a K(m) of 94 micromolar. The molecular weight of the enzyme from Chlamydomonas was estimated to be 92,000 by gel filtration.The phosphatase from both sources required a divalent cation for activity. The Chlamydomonas enzyme was most effectively activated by Co(2+), but was also activated by Mg(2+) (K(a) = 30 micromolar), Mn(2+), and Zn(2+). The Anacystis enzyme was most effectively activated by Mg(2+) (K(a) = 140 micromolar), and was also activated by Co(2+) and Mn(2+), but not by Zn(2+). Anions were also required for maximum activity of the enzyme from both sources. The Chlamydomonas enzyme was activated about 2- to 3-fold by chloride (K(a) = 140 micromolar), bromide, nitrate, bicarbonate (K(a) = 600 micromolar) and formate. The Anacystis enzyme was activated over 10-fold by chloride (K(a) = 870 micromolar), bromide, iodide, and nitrate, but was not activated by bicarbonate or formate.The properties of the algal enzymes were similar to those previously reported for higher plants. The levels and kinetic properties of the enzyme seemed sufficient to account for the flux through the glycolate pathway that occurs in these algae. The phosphatase was not associated with the ribulose 1,5-bisphosphate carboxylase/oxygenase responsible for P-glycolate formation in the carboxysomes of Anacystis.

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii.

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO(2) is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO(2) required for the half-maximal rate of photosynthetic O(2) evolution (K(0.5)[CO(2)]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO(2) fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K(0.5)(CO(2)) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO(2) is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO(2) from HCO(3) (-) at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO(3) (-) transport for internal accumulation might occur at the level of the chloroplast envelope.

Circulatory clearance, internalization, and degradation of muscle AMP aminohydrolase.

Chicken muscle AMP aminohydrolase is cleared rapidly from the circulation of chickens after intravenous injection of the purified enzyme (Husic, H. D., and Suelter, C. H. (1980) Biochem. Biophys. Res. Commun. 95, 228-235). After the intravenous injection of unlabeled, 125I-labeled, or [14C]sucrose-labeled AMP aminohydrolase, enzyme activity and radioactivity are cleared at the same rates and concentrate in the liver and spleen. After injection of the [14C]sucrose-labeled enzyme, 14C is retained in the liver and spleen and low molecular weight 14C is recovered primarily in a fraction which cosediments with lysosomes when tissue homogenates are sedimented on sucrose density gradients. When liver cells are fractionated after clearance of [14C]sucrose-labeled enzyme, 14C is recovered primarily in the parenchymal cells. The circulatory clearance of AMP aminohydrolase is inhibited by heparin and other sulfated polysaccharides, but not by compounds which inhibit previously described carbohydrate-mediated systems for the uptake of circulatory glycoproteins. Heparin injection after the clearance of AMP aminohydrolase causes the release of the enzyme from the liver and spleen into the circulation. When heparin is injected at various times after clearance, decreasing amounts of enzyme are released with time; these results show that the enzyme is internalized with a half-life of 0.98 h.

Binding, internalization, and degradation of AMP aminohydrolase by avian hepatocyte monolayers.

Chicken muscle AMP aminohydrolase is cleared from the circulation of chickens after intravenous injection of the purified enzyme with a half-life of 3-5 min (Husic, H.D., and Suelter, C.H. (1980) Biochem. Biophys. Res. Commun. 95, 228-235). The enzyme is not inactivated before clearance, the clearance is inhibited by sulfated polysaccharides, and the enzyme is cleared primarily by the spleen and the parenchymal cells of the liver where it is internalized and degraded in lysosomes (Husic, H.D., and Suelter, C.H. (1984) J. Biol. Chem. 259, 4359-4364). The binding of AMP aminohydrolase to hepatocyte monolayers in vitro at 4 degrees C is saturable with a dissociation constant of 11.3 X 10(-8) M; there are 2.6 X 10(6) AMP aminohydrolase binding sites/hepatocyte. The interaction of the enzyme with hepatocyte monolayers is inhibited by sulfated polysaccharides, effectors of its enzymatic activity and high salt concentrations; various monosaccharides had little effect on the binding of the enzyme to hepatocyte monolayers. Heparitinase treatment of hepatocyte monolayers abolished 77% of the binding of the enzyme. Heparin promotes the dissociation of 125I-labeled or [14C]sucrose-labeled enzyme bound to the cell surface; radioactivity which is not dissociated by heparin is assumed to be internalized at 37 degrees C. Low molecular weight 125I-labeled degradation products are released into the media with time when the 125I-labeled enzyme, bound to hepatocytes at 4 degrees C, is incubated at 37 degrees C; when [14C]sucrose-labeled enzyme is incubated with hepatocytes at 37 degrees C, low molecular weight 14C-labeled degradation products are not released into the media but instead accumulate in the cells. The half-life for internalization of the bound enzyme based on this rate of accumulation is 0.77 h. These results suggest that glycosaminoglycans are involved in the binding of AMP aminohydrolase to the hepatocyte cell surface and that the bound enzyme is internalized and degraded.

Anion and divalent cation activation of phosphoglycolate phosphatase from leaves.

Phosphoglycolate (P-glycolate) phosphatase was purified 223-fold from spinach leaves by (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and Sephadex G-200 chromatography. The partially purified enzyme had a broad pH optimum between 5.6 and 8.0 and was specific for the hydrolysis of P-glycolate with a Km (P-glycolate) of 26 microM. The enzyme was activated by divalent cations including Mg2+, Co2+, Mn2+, and Zn2+, and by anions including Cl-, Br-, NO-3, and HCOO-. Neither anions nor divalent cations activated the enzyme without the other. The P-glycolate phosphatase activities from tobacco leaves or the green algae, Chlamydomonas reinhardtii, also required Mg2+ and were activated by chloride. In addition, the enzyme was allosterically inhibited by ribose 5-phosphate. The activation of P-glycolate phosphatase by both anions and divalent cations and the inhibition by ribose 5-phosphate may be involved in the in vivo regulation of P-glycolate phosphatase activity.

The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation.

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.