Chloroplast structure and function in ac-20, a mutant strain of Chlamydomonas reinhardi. II. Photosynthetic electron transport.

Photosynthetic electron transport is markedly affected in mixotrophic cells of ac-20 because they lack the capacity to form the wild-type level of cytochrome 559, as well as Q, the quencher of fluorescence of photochemical system II. The other components of the electron-transport chain, as well as reactions dependent upon photochemical system I, are unaffected in the mutant strain. These observations are discussed in terms of the previously reported effects of the ac-20 mutation on CO(2) fixation and ribulose-1,5-diphosphate carboxylase activity.

Synthesis of chloroplast membrane polypeptides during synchronous growth of Chlamydomonas reinhardtii.

The synthesis of the major chloroplast membrane polypeptides has been studied during synchronous growth of Chlamydomonas reinhardtii. Under these conditions, chlorophyll is synthesized during the latter part of the light period and cell division takes place during the dark period. The profile of the chloroplast membrane polypeptides of C. reinhardtii has been well characterized and shown to contain two major classes by size (Hoober, J. 1970. J. Biol. Chem.245:4327). Polypeptides of group I have a mol wt range of 50,000-55,000 daltons. The second region consists of at least three polypeptide groups, IIa, IIb, and IIc, having mol wt of 40,000, 31,000, and 27,000 daltons, respectively. The synthesis of these polypeptides has been measured using a double-labeling technique and a computer-aided statistical analysis. The rate of labeling of group I polypeptides is highest during the early light period and decreases after 6 h of growth. Group IIa is labeled from the beginning of the light period, but little synthesis of IIb occurs before 3 h, and significant amounts of label are not found in IIc before 5 h of growth. After approximately 8 h of light, groups IIb and IIc are synthesized at rates significantly greater than those of the other membrane polypeptides. The synthesis of the major polypeptide groups ceases in the dark. We conclude that the biosynthesis of the chloroplast membranes is a sequential or stepwise process.

Chloroplast structure and function in ac-20, a mutant strain of Chlamydomonas reinhardi. I. CO2 fixation and ribulose-1,5-diphosphate carboxylase synthesis.

A mutant strain of the green alga Chlamydomonas reinhardi, ac-20, is described in which both the rate of CO(2) fixation by whole cells and the rate of carboxylation of ribulose-1,5-diphosphate in cell-free extracts are reduced, particularly when sodium acetate is present in the growth medium. Of the enzymes of the reductive pentose phosphate cycle tested, only ribulose-1,5-diphosphate carboxylase activity is reduced in the mutant strain, and it appears that the low carboxylase activity limits the strain's rate of photosynthetic carbon metabolism. Evidence is presented to show that the fluctuation in the level of the enzyme activity in the presence or absence of acetate results from the fluctuation in the level of some factor(s) limiting the rate of synthesis of the protein.

The effects of inhibitors of RNA and protein synthesis on the recovery of chloroplast ribosomes, membrane organization, and photosynthetic electron transport in the ac-20 strain of Chlamydomonas reinhardi.

The ac-20 strain of Chlamydomonas reinhardi is characterized by low levels of chloroplast ribosomes when grown mixotrophically. Cells can be transferred to minimal medium and their ribosome levels increase. If, at the time of transfer, cells are exposed to chloramphenicol, an inhibitor of protein synthesis in the chloroplast, or cycloheximide, an inhibitor of protein synthesis in the cytoplasm, ribosome recovery is not affected; however, recovery is blocked by exposure to rifampicin, an inhibitor of chloroplast DNA-dependent RNA polymerase. It is therefore concluded that ac-20 cells suffer from an impaired chloroplast ribosomal RNA synthesis. Mixotrophic ac-20 cells are also characterized by low rates of photosynthetic electron transport, disorganized chloroplast membranes, and a small pyrenoid. If chloramphenicol is applied to transferred cells whose chloroplast ribosome levels have already recovered, recovery of photosynthetic electron transport and of structural integrity does not occur. Under the same conditions, cycloheximide has no effect on recovery. It is concluded that the structural and photosynthetic lesions in ac-20 are a secondary consequence of the low levels of chloroplast ribosomes. Finally, we present evidence that recovery of photosynthetic electron transport requires the transcription of chloroplast DNA. This transcription is apparently triggered by light.

Chloroplast structure and function in ac-20, a mutant strain of Chlamydomonas reinhardi. 3. Chloroplast ribosomes and membrane organization.

The fine structure of the ac-20 strain of Chlamydomonas reinhardi is described. Cells grown mixotrophically in the presence of acetate have a highly disordered chloroplast membrane organization and usually lack pyrenoids. Chloroplast ribosome levels are only 5-10% of wild-type levels. Cells grown phototrophically without acetate possess more chloroplast ribosomes and have more normal membrane and pyrenoid organization. Chloroplast ribosome levels rise rapidly when cells are transferred from acetate to minimal medium, whereas membrane reorganization occurs only after a lag. These results, combined with earlier studies of the photosynthetic properties of the mutant strain, suggest that proper membrane organization, Photosystem II activity, and ribulose-1,5-diphosphate carboxylase formation are dependent on the presence of chloroplast ribosomes. Other chloroplast components tested are unaffected by a 10-fold reduction in levels of chloroplast ribosomes.

Synthesis of chloroplast membrane lipids and chlorophyll in synchronous cultures of Chlamydomonas reinhardi.

Chloroplast membrane lipid synthesis has been studied in synchronously growing cultures of Chlamydomonas reinhardi. The synthesis of sulfolipid and phospholipid were measured by incorporation of 35SO4(2-) and 32PO4(3-) during a 1-h pulse. Galactolipid synthesis was measured by H14CO3- incorporation into lipid fractions separated by thin layer chromatography. Lipid synthesis occurs principally during the light portion of the synchronous cycle. Phosphatidylglycerol is synthesized between 3-4 h in the light and sulfolipid is labeled between 7-9 h in the light. Galactolipid synthesis appears to reach maximal rates shortly after the lights go on and again at 7 h. Chlorophyll reaches maximal rates of synthesis after 7 h. These lipids are made and inserted into the chloroplast membrane prior to major increases in photosynthetic capacity. Our results also show that chloroplast membrane lipids are synthesized in a sequential or multistep process.

Membrane polypeptides of some higher plant chloroplasts.

Sodium dodecylsulfate-polyacrylamide gel electrophoresis of membrane polypeptides of the mesophyll cell chloroplasts of barley, pea, and maize show similar profiles, with the polypeptides falling into two major groups: those associated with a membrane fraction enriched in Photosystem I (called Group I polypeptides) and those associated with a membrane fraction enriched in Photosystem II (called Group II polypeptides a, b, and c). In contrast to these profiles, the polypeptides from the extensively unstacked membranes of chloroplasts from the chlorophyll-deficient mutant strains of barley and pea as well as those obtained from the agranal bundle sheath cell chloroplasts of maize are deficient in the Group II polypeptides b and c. It is proposed that these polypeptides are required for membrane stacking in higher plant chloroplasts. These Group II polypeptides b and c are not required for Photosystem II activity since both the barley and pea mutant chloroplasts and the maize bundle sheath chloroplasts possess Photosystem II activities.

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor o.

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission. Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights 0f 32 000, 23 000 and 15 000. The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF1-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark difference in labeling are observed in CF1-depleted chloroplasts which have been treated with N,N'-dicyclohexylcarbodiimide. The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CFo) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CFo, and the light/dark labeling differences may indicate conformational changes within CFo. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.

The esterase-like activity of covalently bound human third complement protein.

The acyl ester bond between the third complement protein, C3, and a variety of molecules is hydrolyzed spontaneously at neutral pH (Venkatesh et al., 1984). Modification of the free, single sulfhydryl group of bound C3 by thiol reagents suggested that a functional group other than the -SH acts as a "catalytic" group in this intramolecular hydrolytic reaction. Complete inhibition of the esterase-like activity is observed with stoichiometric amounts of mercuric chloride, palladium chloride, and the bifunctional organic mercurial, 3,6-bis-(acetoxymercuri)-o-toluidine [BAMT]. Since alkyl and aryl mercuric ions do not inhibit the esterase-like activity of C3-[3H]glycerol, it is conjectured that divalent mercury, palladium, and BAMT will form a complex with the -SH group and an atom of the "catalytic" group X having a lone pair of electrons. The structural features of C3 that are essential for the esterase-like activity remain intact after subjecting C3-[3H]glycerol to covalent chromatography on organomercurial agarose. Based on the observed effects of chemical reagents and the kinetic deuterium solvent isotope effect on the esterase-like activity, a general-base mechanism is proposed for the intramolecular hydrolysis of the acyl ester bond in covalently bound C3. The "catalytic" group X is located in the C3d region (residues 317-632 of the alpha chain), since C3d-[3H]glycerol also has esterase-like activity. A general-base mechanism mediated by the same "catalytic" group X may also apply to the formation of acyl ester bonds following the hydrolysis of the internal thiolester bond in native C3.

Evidence showing that the 1105 and 1106 isotypic residues of the fourth component of human complement, C4A, are not involved in amide bond formation.

Human C4A and C4B have different functions that may stem from their ability to bind hydroxyl or free amino groups on complement activating surfaces. Previous studies suggest that C4B binds to hydroxyl or amino groups whereas C4A binds to free amino groups on acceptor molecules. Comparison of the derived amino acid sequences of C4A and C4B has shown that differences exist between them at positions 1101, 1102, 1105 and 1106. These residues appear to be involved in the binding specificity of C4B. Less is known about the corresponding residues of C4A. It has been suggested that the aspartic acid of C4A at position 1106 is involved in amide bond formation by serving as a catalytic residue for the reaction or by promoting an increased interaction with amino nucleophilic groups. To examine the functional role of residues 1101-1106, we studied the effects of the C4A site-specific antipeptide mAb, AII-1 in assays dependent on the covalent binding properties of C4A; the C4 mediated inhibition of hemolysis and the C4 mediated inhibition of immune precipitation. This study shows that mAb AII-1 has no effect on C4-mediated hemolysis or its ability to inhibit the rate of immune precipitate formation. The lack of interference by AII-1 in these assays could not be explained by low affinity interaction between antibody and C4A showing that mAb AII-1 does not affect the covalent binding activity of C4A. Furthermore, results from epitope mapping studies show that AII-1 binds to Leu1105 and Asp1106 suggesting that these residues are not critical for amide bond formation by C4A.

The fluid-phase binding of human C4 and its genetic variants, C4A3 and C4B1, to immunoglobulins.

Covalent binding of the fourth complement protein, C4, to immune complexes is an important first step in the complement mediated processing of the complexes. Many of the initial encounters between the proteins of the complement system and antigen and antibody occur in solution, and prior to this report, studies of the interactions between them have focused on complement binding to preformed immune precipitates that most likely are not found in vivo. We have characterized the covalent binding of C4b to immunoglobulin molecules in a fluid-phase system consisting only of antibody in solution and purified C4 and C1s. We demonstrate that human C4b binds to IgG in the fluid phase, that its covalent binding is predominantly to the heavy chain of IgG, and that the covalent linkage is by either amide or acyl ester bonds. In addition, we compare the covalent binding efficiencies of two genetic variants of C4, C4A3 and C4B1, to IgG. C4A3 binds 3-4 times more IgG than C4B1 over a range of C4 concentrations, and C4A3 has a higher binding efficiency than C4B1 for IgM, IgA, IgG2a and F(ab')2 as well as for a protein antigen, BSA. Furthermore, we found that whereas C4A3 is bound to immunoglobulins in the fluid-phase predominantly by amide linkage, C4B1 is bound by either amide or acyl ester bonds. The results presented here suggest that the covalent binding efficiency of C4A3 and C4B1 to IgG is similar to that reported for their covalent binding to small molecules.

Synthetic peptide inhibitors of complement serine proteases--I. Identification of functionally equivalent protease inhibitor sequences in serpins and inhibition of C1s and D.

Sequence homology comparisons between serum serine protease inhibitors led to the prediction that the C-terminal sequences are functionally equivalent and represent an essential protease binding domain. Inhibition of complement serine protease D cleavage of factor B and of C1s cleavage of C4 by synthetic peptides containing sequences from the C-termini of three serum serine protease inhibitors supports this prediction. These functionally equivalent peptides represent a new class of inhibitors of D and C1s as well as other serum serine proteases.

Synthetic peptide inhibitors of complement serine proteases--III. Significant increase in inhibitor potency provides further support for the functional equivalence hypothesis.

Synthetic peptides based on functionally equivalent (as defined by similar patterns of chemically equivalent amino acids) serine protease inhibitor (serpin) C-terminal sequences inhibit both classical and alternative pathways of complement activation. Inhibition was also found with hybrid peptides consisting of the cleavage site of one serpin (antithrombin III, alpha-1-antitrypsin, or antichymotrypsin) attached to the short and long functionally equivalent protease binding cores of the other two serpins. A hybrid peptide composed of the sequence at the site of cleavage of C4 by C1s attached to the long binding core of antithrombin III was selective in inhibiting the classical pathway with no effect on the alternative pathway at a concn of 10 microM. Extension of the functional equivalence hypothesis has produced inhibitors of complement activation named generic and generic +, whose sequences differ by 77% or 87%, respectively, from those of all three serpin sequences. A hybrid peptide composed of the antithrombin III cleavage site attached to the generic peptide is an inhibitor of complement activation at 500 nM, the most potent inhibitor found in this study.

Synthetic peptide inhibitors of complement serine proteases--II. Effects on hemolytic activity and production of C3a and C4a.

Synthetic peptides based on the amino acid sequence at the site of cleavage of C3 by classical and alternative pathway convertases were found to be poor inhibitors of hemolysis except at concns of 1 mM and higher. Synthetic peptides of a second type, based on the C-terminal sequence of antithrombin III, were more effective; the best among them caused significant inhibition of hemolysis at a concn of 5 microM. A hybrid peptide composed of the sequence at the site of cleavage of C4 by C1s attached to an antithrombin III sequence was selective, inhibiting the classical pathway with no effect on the alternative pathway at a concentration of 25 microM. Several of the antithrombin III peptides that inhibited hemolysis did not inhibit C4 activation by the classical pathway or activation of C3 by the classical and alternative pathways suggesting that these peptides affect hemolysis by inhibiting enzymes other than C1s and C4b2a of the classical pathway and C3bBb of the alternative pathway.

Isolation and initial characterisation of complement components C3 and C4 of the nurse shark and the channel catfish.

Complement components C3 and C4 have been isolated from the serum of the nurse shark (Ginglymostoma cirratum) and of the channel catfish (Ictalurus punctatus). As in the higher vertebrates, the fish C4 proteins have three-chain structures while the C3 proteins have two-chain structures. All four proteins have intra-chain thioesters located within their highest molecular mass polypeptides. N-terminal sequence analysis of the polypeptides has confirmed the identity of the proteins. In all cases except the catfish C3 alpha-chain, which appears to have a blocked N-terminus, sequence similarities are apparent in comparisons with the chains of C3 and C4 from higher vertebrates. We have confirmed that the activity/protein previously designated C2n is the nurse shark analogue of mammalian C4. This is the first report of structural evidence for C4 in both the bony and cartilaginous fish.

IgM rheumatoid factor and the inhibition of covalent binding of C4b to IgG in immune complexes.

Work by other investigators has shown that IgM-rheumatoid factors (IgM-RF's) can impede complement-mediated inhibition of immune precipitation. We examined the binding of complement component C4b to radiolabelled IgG in model immune complexes and demonstrate that IgM-RF's are capable of reducing the covalent binding of C4b to 125I-IgG in the complexes. Reduced binding to IgG, however, may not be accompanied by binding of C4b to IgM-RF's within the complex, as we also demonstrate that IgM-RF's are relatively poor at C4b capture compared with normal IgM.