Claude D. Stringer

Purification and characterization of ribulose-5-phosphate kinase from spinach

An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.

Complete primary structure of ribulosebisphosphate carboxylase/ oxygenase from Rhodospirillum rubrum

Of the 14 cyanogen bromide fragments derived from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, four are too large to permit complete sequencing by direct means [F. C. Hartman, C. D. Stringer, J. Omnaas, M. I. Donnelly, and B. Fraij (1982) Arch. Biochem. Biophys. 219, 422-437]. These have now been digested with proteases, and the resultant peptides have been purified and sequenced, thereby providing the complete sequences of the original fragments. With the determination of these sequences, the total primary structure of the enzyme is provided. The polypeptide chain consists of 466 residues, 144 (31%) of which are identical to those at corresponding positions of the large subunit of spinach ribulosebisphosphate carboxylase/oxygenase. Despite the low overall homology, striking homology between the two species of enzyme is observed in those regions previously implicated at the catalytic and activator sites.

[83] Ribulosebisphosphate car☐ylase/oxygenase from Rhodospirillum rubrum

Publisher Summary This chapter describes the assay method, the purification procedure, and the properties of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum . Ribulosebisphosphate carboxylase activity is determined spectrophotometrically at 340 nm. Oxidation of NADH is monitored during the conversion of 3-phosphoglycerate, the product of the carboxylation reaction, to glycerol 3-phosphate as catalyzed in concert by 3-phosphoglycerate phosphokinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and glycerophosphate dehydrogenase. The isolation and purification process involves preparation of cell suspension, diethylaminoethyl (DEAE)-cellulose chromatography, and second DEAE-cellulose chromatography. The pure enzyme sediments with a S 20,w of 6.2 S and its molecular weight—as determined by light scattering—is 114,000. Its subunit molecular weight as judged by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) is 56,000. Like all ribulosebisphosphate carboxylase/oxygenases, the R. rubrum enzyme is devoid of both activities in the absence of divalent metal ions and CO 2 . Many phosphate esters and other di- or multivalent anions competitively inhibit the R. rubrum carboxylase; however, the inhibition is generally weaker than that observed for the corresponding plant enzymes.

Sequences of two active-site peptides from spinach ribulosebisphosphate carboxylase/oxygenase☆

Abstract Two tryptic peptides from spinach ribulosebisphosphate carboxylase/oxygenase that contain the essential lysyl residues derivatized by the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate were subjected to sequence analyses. The sequences of these peptides are -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys-Thr-Ile-Lys-Pro-Lys- and -Leu-Ser-Gly-Gly-Asp-His-Ile-His-Ser-Gly-Thr-Val-Val-Gly-Lys-Leu-Glu-Gly-Glu-Arg-, respectively. The reagent moiety is covalently attached to the internal lysyl residue in each peptide.

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Abstract Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [ 3 H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys ∗ Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys ∗ AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys ∗  ThrThrAsxAsxPheThrArg AlaCys ∗ ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys ∗ HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.

Purification and sequencing of cyanogen bromide fragments from ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum☆

Abstract As a part of the goal to determine the total sequence of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, the cyanogen bromide fragments were fractionated and sequenced (or partially sequenced). Twelve of the anticipated 14 peptides were obtained in highly purified form. The other two peptides were located, respectively, within a trytophanyl cleavage product (which overlapped with four CNBr fragments) and within an active-site peptide characterized earlier (which overlapped with three CNBr fragments). These overlaps coupled with amino and carboxyl terminal sequence information of the intact subunit and the availability of the sequence of the corresponding enzyme from higher plants permitted alignment of all fragments. Eight CNBr peptides were sequenced completely; four of the CNBr peptides consisted of more than 80 residues and were only partially sequenced as permitted by direct Edman degradation. Of the approximate 475 residues per subunit, 339 were placed in sequence. The lack of extensive conservation of primary structure between R. rubrum and higher plant carboxylases permits the tentative identifications of those regions likely to be functionally important.

Characterization of an active-site peptide modified by glyoxylate and pyridoxal phosphate from spinach ribulosebisphosphate carboxylase/oxygenase.

Activated ribulosebisphosphate carboxylase/oxygenase from spinach was treated with glyoxylate plus or minus the transition-state analog, carboxyarabinitol bisphosphate, or the inactive enzyme with pyridoxal phosphate plus or minus the substrate, ribulose bisphosphate. Covalently modified adducts with glyoxylate or pyridoxal phosphate were formed following reduction with sodium borohydride. The derivatized enzymes were carboxymethylated and digested with trypsin; the labeled peptides which were unique to the unprotected samples were purified by ion-exchange chromatography and gel filtration. Both glyoxylate and pyridoxal phosphate were associated with only one major peptide, which in each case was subjected to amino acid analysis and sequencing. The sequence was -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys(Cm)-Thr-Ile-Lys-Lys*-Pro-Lys-, with both reagents exhibiting specificity for the same lysine residue as indicated by the asterisk. This peptide is identical to that previously isolated from spinach carboxylase labeled with either of two different phosphorylated affinity reagents and homologous to one from Rhodospirillum rubrum carboxylase modified by pyridoxal phosphate. The species invariance of this lysine residue, number 175, and the substantial conservation of adjacent sequence support the probability for a functional role in catalysis of the lysyl epsilon-amino group.