Molecular evolution of C4 phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase in the genus<Emphasis Type="Italic"> Flaveria</Emphasis>—a gradual increase from C3 to C4 characteristics

In order to elucidate the discrete steps in phospho enolpyruvate carboxylase (PEPC) evolution concerning K(m)-PEP and malate tolerance a comparison was made between C3, C3-C4 and C4 species of the dicot genus Flaveria. The PEPCs of this genus are encoded by a gene family comprising three classes: ppcA, ppcB and ppcC [J. Hermans and P. Westhoff (1990) Mol Gen Genet 224:459-468, (1992) Mol Gen Genet 234:275-284]. The ppcA of F trinervia (C4) codes for the C4 PEPC isoform but other plants of the genus contain ppcA orthologues too. The C3 plant F. pringlei showed the lowest levels of ppcA PEPC mRNA followed by F. pubescens (C3-C4) while the C4-like plant F. brownii displayed RNA amounts close to the C4 species F. trinervia. In contrast to the similar expression profiles of F. brownii (C4-like) and F. trinervia (C4) the PEPC amino acid sequence of F. brownii was more similar to the C3 and C3-C4 ppcA PEPCs than to the C4 PEPC. Similarly, the C3, C3-C4 and C4-like ppcA PEPCs showed almost identical PEP saturation kinetics when activated by glucose-6-phosphate ( K(m)-PEP: 17-20 microM) while the K(m)-PEP for the C4 PEPC was determined to be 53 microM. However, without activation the ppcA PEPCs of F. pubescens and F. brownii displayed C3-C4 intermediate values. A similar picture was obtained when the malate sensitivities were compared. In the non-activated state the F. trinervia (C4) enzyme was 10 times more tolerant to malate than the F. pringlei counterpart. The ppcA enzymes of F. pubescens (C3-C4) and F. brownii (C4-like) displayed intermediate values. In contrast, the inclusion of 5 mM glucose-6-phosphate in the reaction mixture changed the order totally. Interestingly, the activation rendered the C4 enzyme about 50% less tolerant to malate than the C3 PEPC. The activation had a positive effect on malate tolerance of the F. pubescens (C3-C4) PEPC while the ppcA PEPC of F. brownii (C4-like) was almost unaffected.     

Subunit polypeptide composition of rubisco and the origin of allopolyploid Arabidopsis suecica (Brassicaceae)

The polypeptide composition of the large and small subunits of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) from Arabidopsis thaliana, A. suecica and Cardaminopsis arenosa have been studied by IEF (isoelectric focusing) analysis. The putative recent alopolyploid origin of A. suecica is supported. The chloroplast encoded large subunits served to identify solely A. thaliana as the maternal parent whereas the nuclear encoded small subunits indicate C. arenosa as the paternal species.     

Subunit C of the vacuolar H+-ATPase of Hordeum vulgare.

The molecular cloning of the first subunit C of the plant vacuolar H+-ATPase is reported. Tonoplast vesicles were purified from barley leaves by sucrose gradient centrifugation, and the tonoplast polypeptides were separated by two-dimensional (2-D) gel electrophoresis. Using an anti-ATPase holoenzyme antibody, a polypeptide was recognized in the molecular mass range of 40 kDa with an isoelectric point of about 6.0, and tentatively identified as subunit C. The polypeptide spot was excised from about 50 2-D gels and subjected to endo Lys C proteolysis. Two proteolytic peptides were sequenced and the amino acid sequences were used to design degenerated oligonucleotides, followed by PCR amplification with cDNA template and screening of a cDNA library synthesized from Hordeum vulgare poly A mRNA of epidermis strips. The full length clone of 1.5 kbp contains an open reading frame of 1062 bp encoding a polypeptide of 354 amino acids with a molecular mass of 39,982 Da and an isoelectric point of 6.04. Amino acid identity with sequences of SUC from animals and fungi is in the range of 36.7 to 38.5%. Expression of the cloned gene was demonstrated by Northern blotting and RT-PCR.     

Photosynthetic Characteristics of Segregates from Hybrids between Flaveria brownii (C4 Like) and Flaveria linearis (C3-C4)

Characteristics related to C4 photosynthesis were studied in reciprocal F1 hybrids and F2 plants from Flaveria brownii (C4 like) and Flaveria linearis (C3-C4). The reciprocal F1 plants differed in 13C/12C ratios of leaves and the percentage of 14C initially incorporated into C4 acids, being more like the pollen parents in these traits. They did not differ in apparent photosynthesis or in O2 inhibition of apparent photosynthesis and differed only slightly in CO2 compensation concentration at 175 [mu]mol quanta m-2 s-1 and 400 mL L-1 O2. The 13C/12C ratios of 78 F2 progeny from the two F1 plants exhibited a normal distribution centered between those of the parents, with a few values slightly higher and lower than the parents. Apparent photosynthesis at 130 [mu]L L-1 CO2 and inhibition of photosynthesis by O2 was nearly normally distributed in the F2 population, but no values for F2 plants approached those for F. brownii (15.4 [mu]mol m-2 s-1 and 7.8%, respectively). Distribution of the CO2 compensation concentration measured at 1000 [mu]mol quanta m-2 s-1 and 400 mL L-1 of O2 in the F2 population was skewed toward F. brownii with 72% of the progeny having values <9 [mu]L of CO2 L-1 compared to 1.5 and 27.2 [mu]L L-1 for F. brownii and F. linearis, respectively. Correlations among traits of F2 plants were low (coefficients of 0.30 to -0.49), indicating that the C4- related traits are not closely linked in segregating populations. Plants in the F2 population selected for high or low apparent photosynthesis at 130 [mu]L of CO2 L-1 (six each) did not rank consistently high or low for 13C/12C ratios, O2 inhibition of apparent photosynthesis, CO2 compensation concentration, or activities of phosphoenolpyruvate carboxylase or NADP-malic enzyme. This study confirms results of earlier work that indicates independent segregation of C4 traits and also shows that the C4-like parental type can be recovered, at least for some characteristics (13C/12C ratio), in segregating populations. Recovery of fully functional C4 plants awaits further experimentation with C4 x C3 or C4 x C3-C4 hybrid plants that produce fertile progeny.     

Analysis of catalytic subunit microheterogeneity in ribulosebisphosphate carboxylase/oxygenase from Nicotiana tabacum

Urea isoelectric focusing of dissociated, carboxymethylated Nicotiana tabacum ribulose-1,5-bisphosphate carboxylase/oxygenase reveals catalytic subunit microheterogeneity. Aggregated or nonaggregated sucrose gradient-purified preparations and the crystalline protein displayed essentially identical large subunit multiple polypeptide patterns. Various pretreatments which fully dissociate the holoenzyme did not alter catalytic subunit microheterogeneity. Direct comparison of the carboxymethylated and noncarboxymethylated crystalline and sucrose gradient-purified proteins demonstrated that the large subunit multiple polypeptide pattern was not an artifact of carboxymethylation. The inclusion of the seryl protease inhibitor phenylmethylsulfonyl fluoride during purification of the holoenzyme did not affect the large subunit multiplicity. However, the addition of leupeptin, a potent thiol proteinase inhibitor, to all solutions during purification of the native protein markedly reduced large subunit polypeptide L₃ and increased the staining of polypeptide L₂, suggesting that L₃ is a leupeptin-sensitive proteinase degradation product of L₂. Polypeptide L₁ also appeared to be a purification-related artifact, but derived from a modification of L₂ other than that which yielded L₃. We conclude that polypeptide L₂ is the single, native isoelectric form of the catalytic subunit of tobacco ribulosebisphosphate carboxylase/oxygenase.     

Formation and properties of flavoprotein-cytochrome hybrids by recombination of subunits from different species.

p-Cresol methylhydroxylases from four different pseudomonads differ in their isoelectric points and, to a lesser extent, in Mr values and substrate specificity. The enzymes from three species were isolated in homogeneous form, then resolved into their flavoprotein and cytochrome subunits, and the subunits were recombined to yield the nine possible hybrids (i.e. three intraspecies and six interspecies). The resulting flavocytochromes showed extensive similarities in steady-state kinetic parameters and in the dissociation constants of their subunits. Evidence is also presented that a fourth type of p-cresol methylhydroxylase, from Pseudomonas putida (N.C.I.B. 9869, form 'B'), the subunits of which cannot be isolated by the isoelectric focusing technique used to separate the subunits of the other flavocytochromes, nevertheless dissociates slowly at high dilution. The dissociation is reflected by a decline of catalytic activity with time. This process for the 'B' enzyme is prevented by the presence of substrate or an excess of a cytochrome subunit isolated from another enzyme species. Incubation of the dissociated subunits with p-cresol brings about extensive, albeit incomplete, re-association and regeneration of activity.     

Ribulose Bisphosphate Carboxylase from Three Chlorophyll c-Containing Algae : Physical and Immunological Characterizations

Distinctive properties are identified in the molecular structure of ribulose, 1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in chlorophyll c-containing algae (i.e., chromophytes). Using purified enzyme from Cryptomonas sp., Coccolithophora sp., and Cylindrotheca fusiformis, we have determined that the RuBPCase holoenzyme of each species has a molecular weight, subunit composition, and isoelectric points of its subunits similar to the purified enzymes from pea and Chlamydomonas reinhardtii. The large subunits from chromophytes exhibit microheterogeneity in their isoelectric points, whereas two to four well-resolved isoelectric variants of the small subunit were observed in each RuBPCase preparation. In spite of the high degree of similarity in terms of physical properties, both the small and large RuBPCase subunits of the chromophytes are structurally different from those of chlorophytes; immunological studies demonstrate that RuBPCase subunits of these two groups have few antigenic determinants in common.     

Evolution of c4 phosphoenolpyruvate carboxylase. Genes and proteins: a case study with the genus Flaveria

C4 photosynthesis is characterized by a division of labour between two different photosynthetic cell types, mesophyll and bundle-sheath cells. Relying on phosphoenolpyruvate carboxylase (PEPC) as the primary carboxylase in the mesophyll cells a CO2 pump is established in C4 plants that concentrates CO2 at the site of ribulose 1,5-bisphosphate carboxylase/oxygenase in the bundle-sheath cells. The C4 photosynthetic pathway evolved polyphyletically implying that the genes encoding the C4 PEPC originated from non-photosynthetic PEPC progenitor genes that were already present in the C3 ancestral species. The dicot genus Flaveria (Asteraceae) is a unique system in which to investigate the molcular changes that had to occur in order to adapt a C3 ancestral PEPC gene to the special conditions of C4 photosynthesis. Flaveria contains not only C3 and C4 species but also a large number of C3-C4 intermediates which vary to the degree in which C4 photosynthetic traits are expressed. The C4 PEPC gene of Flaveria trinervia, which is encoded by the ppcA gene class, is highly expressed but only in mesophyll cells. The encoded PEPC protein possesses the typical kinetic and regulatory features of a C4-type PEPC. The orthologous ppcA gene of the C3 species Flaveria pringlei encodes a typical non-photosynthetic, C3-type PEPC and is weakly expressed with no apparent cell or organ specificity. PEPCs of the ppcA type have been detected also in C3-C4 intermediate Flaveria species. These orthologous PEPCs have been used to determine the molecular basis for C4 enzyme characteristics and to understand their evolution. Comparative and functional analyses of the ppcA promoters from F. trinervia and F. pringlei make it possible to identity the cis-regulatory sequences for mesophyll-specific gene expression and to search for the corresponding trans-regulatory factors.     

The evolution of fraction I protein during the origin of a new species ofNicotiana

The polypeptide composition of Fraction I protein from Nicotiana digluta, a synthetic species which arose by chromosome doubling following the interspecific hybridization of N. glutinosa and N. tabacum, has been examined by isoelectric focusing. The composition of the protein from N. digluta, which was identical to the protein from the infertile F1 hybrid N. glutinosa x N. tabacum, showed 3 polypeptides in the large subunit and 4 polypeptides in the small subunit. The large subunit polypeptides were identical to those from N. glutinosa, the maternal parent in the original hybridization, whereas the small subunit polypeptides were a composite of the small subunit polypeptides from both N. glutinosa and N. tabacum. This analysis demonstrates how the polypeptide composition of Fraction I protein evolves during the origin of new species of Nicotiana.     

Pyruvate carboxylase from a thermophilic Bacillus: some molecular characteristics.

Analysis of the native enzyme and of the subunits produced upon its denaturation shows that pyruvate carboxylase from a thermophilic Bacillus is a tetramer with a molecular weight (mean value) of 558,000 and that the four polypeptide subunits are probably identical. The three functions (carboxyl carrier, carboxylation, and carboxyl transfer) in the pyruvate carboxylation reaction must therefore reside in this quarter-molecular polypeptide. The enzyme molecule contains four atoms of zinc and four molecules of D-biotin, and in the electron microscope the disposition of its four subunits presents a rhombic appearance. Reaction of the denatured enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reveals 10 sulfhydryl groups/subunit. In the native enzyme less than one of these groups reacts with DTNB. By contrast, all of these groups (11/subunit) of the native chicken liver pyruvate carboxylase are accessible to DTNB. The thermophile enzyme is also more resistant to other sulfhydryl reagents and to denaturation under certain conditions than the avian enzyme.     

Separation of PP2A core enzyme and holoenzyme with monoclonal antibodies against the regulatory A subunit: abundant expression of both forms in cells.

Protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit, C, and two regulatory subunits, A and B. The A subunit is rod shaped and consists of 15 nonidentical repeats. According to our previous model, the B subunit binds to repeats 1 through 10 and the C subunit binds to repeats 11 through 15 of the A subunit. Another form of PP2A, core enzyme, is composed only of subunits A and C. It is generally believed that core enzyme does not exist in cells but is an artifact of enzyme purification. To study the structure and relative abundance of different forms of PP2A, we generated monoclonal antibodies against the native A subunit. Two antibodies, 5H4 and 1A12, recognized epitopes in repeat 1 near the N terminus and immunoprecipitated free A subunit and core enzyme but not holoenzyme. Another antibody, 6G3, recognized an epitope in repeat 15 at the C terminus and precipitated only the free A subunit. Monoclonal antibodies against a peptide corresponding to the N-terminal 11 amino acids of the A alpha subunit (designated 6F9) precipitated free A subunit, core enzyme, and holoenzyme. 6F9, but not 5H4, recognized holoenzymes containing either B, B', or B" subunits. These results demonstrate that B subunits from three unrelated gene families all bind to repeat 1 of the A subunit, and the results confirm and extend our model of the holoenzyme. By sequential immunoprecipitations with 5H4 or 1A12 followed by 6F9, core enzyme and holoenzyme in cytoplasmic extracts from 10T1/2 cells were completely separated and they exhibited the expected specificities towards phosphorylase a and retinoblastoma peptide as substrates. Quantitative analysis showed that under conditions which minimized proteolysis and dissociation of holoenzyme, core enzyme represented at least one-third of the total PP2A. We conclude that core enzyme is an abundant form in cells rather than an artifact of isolation. The biological implications of this finding are discussed.     

Functional role of the noncatalytic subunit of complement C5 convertase

The C5 convertase is a serine protease that consists of two subunits: a catalytic subunit which is bound in a Mg2+-dependent complex to a noncatalytic subunit. To understand the functional role of the noncatalytic subunit, we have determined the C5-cleaving properties of the cobra venom factor-dependent C5 convertase (CVF, Bb) made with CVF purified from the venom of Naja naja (CVFn) and Naja haje (CVFh) and compared them to those for two C3b-dependent C5 convertases (ZymC3b,Bb and C3b,Bb). A comparison of the kinetic parameters indicated that although the four C5 convertases (CVFn,Bb, ZymC3b,Bb, CVFh,Bb, and C3b,Bb) had similar catalytic rate constants (kcat = 0.004-0.012 s-1) they differed 700-fold in their affinity for the substrate as indicated by the Km values (CVFn,Bb = 0.036 microM, ZymC3b,Bb = 1.24 microM, CVFh,Bb = 14.0 microM, and C3b,Bb = 24 microM). Analysis of binding interactions between C5 and the noncatalytic subunits (CVFh or C3b, or CVFn) using the BIAcore, revealed dissociation binding constants (Kd) that were similar to the Km values of the respective enzymes. The kinetic and binding data demonstrate that the binding site for C5 resides in the noncatalytic subunit of the enzyme, the affinity for the substrate is solely determined by the noncatalytic subunit and the catalytic efficiency of the enzyme appears not to be influenced by the nature of this subunit.     

Characterization of the estrogen-induced uterine peroxidase by microelectrophoresis

Estrogen-dependent peroxidase from rat uterine fluid has been investigated by microelectrophoretic techniques. The molecular weight of the enzyme has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergent. The isoelectric points are located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, the enzyme was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000. The large subunit has slight enzymatic activiy, while the smaller subunit may be responsible for the charge difference in the holoenzyme pattern. The glycoprotein pattern of the uterine fluid peroxidase is further defined by its separation by affinity chromatography using a concanavalin A-Sepharose column and by its susceptibility to neuraminidase treatment.     

Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits

An expression plasmid incorporating the structural gene for the large subunit of a cyanobacterial ribulose-bisphosphate carboxylase, but not the gene for its complementary small subunit, directs the synthesis of large subunits in Escherichia coli. This provides a means for obtaining a preparation of large subunits completely devoid of small subunits, which is not otherwise achievable. In extracts, these large subunits were found predominantly in the form of octamers, but intersubunit interactions were weaker than in the holoenzyme, which contains eight small subunits as well as eight large subunits, and tended to be broken by procedures which separated octamers from lower oligomers and monomers. However, partial purification by anion-exchange chromatography was possible. The large subunits recognized the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate, thus enabling measurement of catalytic site concentrations, but the binding was much weaker than to the holoenzyme. E. coli-produced large subunits catalyzed carboxylation with a kcat of 1% of that of the holoenzyme and the substrate affinities were 3- to 5-fold weaker. They also assembled with heterologous small subunits isolated from spinach ribulose-P2 carboxylase with a 100-fold increase in catalytic activity under standard assay conditions. Since catalysis can proceed in their absence, the small subunits cannot be directly involved in the catalytic chemistry. Their stimulative influence upon catalysis must be exerted by conformational means.     

Biosynthesis of ribulose-1.5-bisphosphate carboxylase in greening cells of Euglena gracilis

Light induction of chloroplast development in Euglena leads to quantitative changes in the protein composition of the soluble cell part. One major part of these is the observed accumulation of ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBPCase) enzyme (EC 4.1.1.39). As measured by immunoelectrophoresis, a small amount of RuBPCase (about 10^-6 pmol) is present in a dark-grown cell, whereas a greening cell (72h) contains 10–20 pmol enzyme. Both the cytoplasmic and chloroplastic translation inhibitors, cycloheximide and spectinomycin, have a strong inhibitory effect on the synthesis of the enzyme throughout the greening process of Euglena cells. Electrophoretic and immunological analyses of the soluble phase prepared from etiolated or greening cells do not show the presence of free subunits of the enzyme. For each antibiotic-treated greening cell, the syntheses of both subunits are blocked. Our data indicate that tight reciprocal control between the syntheses of the two classes of subunits occurs in Euglena. In particular, the RuBPCase small subunit synthesis in greening Euglena seems more dependent on the protein synthesis activity of the chloroplast than the syntheses of other stromal proteins from cytoplasmic origin.Abbreviations LSU large subunit of ribulose-1.5-bisphosphate carboxylase - RuBP ribulose-1.5-bisphosphate - RuBP-Case ribulose-1.5-bisphosphate carboxylase - SSU small subunit of ribulose-1.5-bisphosphate carboxylase     

The yeast mitochondrial adenosine triphosphatase complex. Subunit stoichiometry and physical characterization

Immunoprecipitation of uniformly labeled yeast submitochondrial preparations using a subunit-specific or a holoenzyme antiserum has been employed to determine the subunit stoichiometry of the oligomycin-sensitive ATPase complex. The Triton-solubilized enzyme consists of 10 types of subunits. The number of copies of each subunit, in order of decreasing molecular weight, is 3:3:1:2:1:2:2:1:2:3. on the basis of the stoichiometry data, the ATPase complex has a molecular weight of 5.8 x 10(5) and contains a minimum of 20 polypeptide chains. Analysis of water-soluble ATPase (F1-ATPase) indicates that the stoichiometry of the three largest subunits of the enzyme is preserved in the absence of the other subunits. The molecular weights of both forms of the ATPase, derived from stoichiometry data, agree well with measurements obtained from gel filtration and sedimentation studies. The implications of these data for the structure, function, and assembly of the complex are discussed.