Biochemistry, Biophysics, and Structural Biology Commons^™

Biotin Carboxyl Carrier Protein And Carboxyltransferase Subunits Of The Multi-Subunit Form Of Acetyl-Coa Carboxylase From Brassica Napus: Cloning And Analysis Of Expression During Oilseed Rape Embryogenesis, Kieran M. Elborough, Robert Winz, Ranjit K. Deka, Jennifer E. Markham, Andrew J. White, Stephen Rawsthorne, Antoni R. Slabas

Department of Biochemistry: Faculty Publications

In the oilseed rape Brassica napus there are two forms of acetyl- CoA carboxylase (ACCase). As in other dicotyledonous plants there is a type I ACCase, the single polypeptide 220 kDa form, and a type II multi-subunit complex analogous to that of Escherichia coli and Anabaena. This paper describes the cloning and characterization of a plant biotin carboxyl carrier protein (BCCP) from the type II ACCase complex that shows 61% identity/79% similarity with Anabaena BCCP at the amino acid level. Six classes of nuclear encoded oilseed rape BCCP cDNA were cloned, two of which contained the entire coding region. …

Elimination Of The Chlamydomonas Gene Family That Encodes The Small Subunit Of Ribulose-1,5-Bisphosphate Carboxylaseyoxygenase, Irina Khrebtukova, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

Ribulose-1,5-bisphosphate carboxylasey oxygenase (EC 4.1.1.39) is the key photosynthetic enzyme that catalyzes the first step of CO₂ fixation. The chloroplastlocalized holoenzyme of plants and green algae contains eight nuclear-encoded small subunits and eight chloroplastencoded large subunits. Although much has been learned about the enzyme active site that resides within each large subunit, it has been difficult to assess the role of eukaryotic small subunits in holoenzyme function and expression. Small subunits are coded by a family of genes, precluding genetic screening or nuclear transformation approaches for the recovery of small-subunit mutants. In this study, the two small-subunit genes of …

Role Of The Escherichia Coli Fadr Regulator In Stasis Survival And Growth Phase-Dependent Expression Of The Uspa, Fad, And Fab Genes, Anne Farewell, Alfredo A. Diez, Concetta C. Dirusso, Thomas Nyström

Department of Biochemistry: Faculty Publications

The increased expression of the uspA gene of Escherichia coli is an essential part of the cell’s response to growth arrest. We demonstrate that stationary-phase activation of the uspA promoter is in part dependent on growth phase-dependent inactivation or repression of the FadR regulator. Transcription of uspA is derepressed during exponential growth in fadR null mutants or by including the fatty acid oleate in the growth medium of FadR1 cells. The results of DNA footprinting analysis show that FadR binds downstream of the uspA promoter in the noncoding region. Thus, uspA is a member of the fadR regulon. All …

In Vivo Regulation Of Wheat-Leaf Phosphoenolpyruvate Carboxylase By Reversible Phosphorylation, Stephen M. G. Duff, Raymond Chollet

Department of Biochemistry: Faculty Publications

Regulation of C₃ phosphoenolpyruvate carboxylase (PEPC) and its protein-serine/threonine kinase (PEPC-PK) was studied in wheat (Trificum aesfivum) leaves that were excised from low-N-grown seedlings and subsequently illuminated and/or supplied with 40 mM KNO₃. The apparent phosphorylation status of PEPC was assessed by its sensitivity to i-malate inhibition at suboptimal assay conditions, and the activity state of PEPC-PK was determined by the in vitro ³²P labeling of purified maize dephospho-PEPC by [y³²PIATP/ Mg. lllumination (±NO₃^-) for 1 h led to about a 4.5-fold increase in the 50% inhibition constant for i-malate, which …

Characteristics Of Modified Leghemoglobins Lsolated From Soybean (Glycine Max Merr.) Root Nodules, Hyung-Kyun Jun, Gautam Sarath, Jose F. Moran, Manuel Becana, Robert V. Klucas, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Hemoprotein derivatives of an abundant soybean (Glycine max Merr.) root nodule leghemoglobin, Lba, were studied for their modified spectral characteristics and physical properties. Three modified hemoprotein derivatives of Lba (Lbam₁, Lbam₂, and Lbam₃) were purified by preparative isoelectric focusing. The ferric forms of these pigments were green and exhibited anomalous spedra in the visible region as compared to the Lba³⁺ forms. These modified pigments showed a hypochromic shift of 10 nm for the charge transfer absorption maximum; however, differences were not apparent in the Sdret region. Upon binding with nicotinate, the α …

Sequence Of A Psac Gene From The Cyanobacterium Synechococcus Sp. Pcc 6301, Patricia L. Herman, Kartika Adiwilaga, John H. Golbeck, Donald P. Weeks

Department of Biochemistry: Faculty Publications

The psaC gene encodes PsaC, the apoprotein for the terminal iron-sulfur clusters, F_A and F_B, in the PSI reaction center of cyanobacteria, algae, and higher plants. PsaC functions as a membrane-bound oxidoreductase, accepting electrons from the F_x iron-sulfur cluster located on the PsaA/PsaB heterodimer and donating them to the soluble electron transfer proteins Fd and flavodoxin (reviewed in Bryant, 1992). The objective of our work is to clarify the role of PsaC in linear and cyclic electron transfer. The experimental organism is Syneckococcus sp. PCC 6301, a unicellular, freshwater cyanobacterium that is used extensively for physiological, …

Exopeptidase Catalyzed Site-Specific Bonding Of Supports, Labels And Bioactive Agents To Proteins, Fred W. Wagner, Thomas R. Coolidge, Dwane E. Wylie, Sheldon M. Schuster, William Lewis, Jay Stout

Department of Biochemistry: Faculty Publications

The invention provides a means for attaching a label, support or bioactive agent to a protein with an exopeptidase at a site that is remote from the active site of the protein. More specifically the invention is directed to a method for the attachment of an amino acid, amine and alcohol nucleophile to the carboxyl terminus of a protein. In one embodiment, a labeled nucleophile is attached to a protein such as an antibody. In other embodiments, the invention is directed to a method for the attachment of a protein to an immobilization support and to a method for the …

Inheritance Of Freezing Resistance In Tuber-Bearing Solanum Species: Evidence For Independent Genetic Control Of Nonacclimated Freezing Tolerance And Cold Acclimation Capacity, Julie M. Stone, Jiwan P. Palta, John B. Bamberg, Laurie S. Weiss, James F. Harbage

Department of Biochemistry: Faculty Publications

Frost or winter survival is regarded as a complex trait with polygenic inheritance. Two maijor components of this survival in crop plants are freezing tolerance in the nonacclimated state and cold acclimation capacity. To date researchers have not distinguished the two components as separate heritable traits. The mode of inheritance of these two traits was investigated in F1 and backcross populations of two wild diploid potato species (Solanum commersonii and Solanum cardiophyllum) exhibiting extremes of freezing tolerance and acclimation capacity. Precise assessment of these two traits allowed distinction of small but significant differences among genotypes. The two traits …

Shared Thematic Elements In Photochemical Reaction Centers, John H. Golbeck

Department of Biochemistry: Faculty Publications

The structural, functional, and evolutionary relationships between photosystem II and the purple nonsulfur bacterial reaction center have been recognized for several years. These can be classified as "quinone type" (type I) photosystems because the terminal electron acceptor is a mobile quinone molecule. The analogous relationship between photosystem I and the green sulfur bacterial (and heliobacterial) reaction centers has only recently become dear. These can be dcassified as "iron-sulfur type" (type I) photosystems because the terminal electron acceptor consists of one or more bound iron-sulfur clusters. At a fundamental level, the quinone type and ironsulfur type reaction centers share a common …

Mutational Analysis Of The Structure And Biogenesis Of The Photosystem I Reaction Center In The Cyanobacterium Synechocystis Sp. Pcc 6803, Lawrence B. Smart, Patrick V. Warren, John H. Golbeck, Lee Mcintosh

Department of Biochemistry: Faculty Publications

We have utilized the unicellular cyanobacterium Synechocystis sp. PCC 6803 to incorporate site-directed amino acid substitutions into the photosystemn I (PSI) reactioncenter protein PsaB. A cysteine residue (position 565 of PsaB) proposed to serve as a ligand to the [4Fe-4S] center F_x was changed to serine, histidine, and aspartate. These three mutants- C565S, C565H, and C565D-all exhibited greatly reduced accumulation of PSI reaction-center proteins and failed to grow autotrophically, indicating that this cysteine most likely does coordinate F_x, which is crucial for PSI biogenesis. Interestingly, the strain C565S accumulated significantly more PSI than the other two cysteine …

Exopeptidase Catalyzed Site-Specific Bonding Of Supports, Labels And Bioactive Agents To Proteins, Fred W. Wagner, Thomas R. Coolidge, Sheldon M. Schuster, Jay Stout, Dwane E. Wylie, Klaus Breddam, William Lewis

Department of Biochemistry: Faculty Publications

An auxiliary substance such as a label, support, or bioactive agent is attached to a protein at a site that is remote from the active site of the protein by the use of exopeptidase and a nucleophile which is an amino acid, amino acid derivative, amine or alcohol. In one embodiment, the nucleophile is attached to the carboxy terminus of a protein by catalysis with exopeptidase to form an adduct and then the adduct or its combination with a linker arm is bound to the auxiliary substance. In another embodiment, the auxiliary substance or its combination with a linker arm …

Chloroplast Heteroplasmicity Is Stabilized By An Amber-Suppressor Tryptophan TrnaCua, Weizhu Yu, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

Photosynthesis-deficient mutants of the green alga Chlamydomonas reinhardtii were previously shown to arise from nonsense mutations within the chloroplast rbcL gene, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Photosynthesiscompetent revertants of these nonsense mutants have often been found to be stably heteroplasmic, giving rise to both mutant and revertant cells during mitotic or meiotic divisions under nonselective growth conditions. It was proposed that the stable heteroplasmicity might arise from a balanced polymorphism of suppressor and wild-type alleles of a tRNA gene maintained within the polyploid chloroplast genome. In the present study, we have focused on determining the …

Lack Of Types 1 And 2a Protein Serine(P)/Threonine(P) Phosphatase Activities In Chloroplasts, Gongqin Sun, John Markwell

Department of Biochemistry: Faculty Publications

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid lightharvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein …

Transition Metals In Legume Root Nodules: Iron-Dependent Free Radical Production Increases During Nodule Senescence, Manuel Becana, Robert V. Klucas

Department of Biochemistry: Faculty Publications

The cytosol from root nodules of soybean, bean, and cowpea contained Fe and Cu capable of catalyzing the formation of highly reactive free radicals. Specific and sensitive assays based on free radical-mediated DNA degradation revealed that most catalytic Fe and Cu were present as small chelates (300-600 Da). The involvement of catalytic Fe in free radical production during nodule senescence, which was induced by exposure of plants to continuous darkness for 2-4 days, was investigated. (i) Free heme remained at a constant and low concentration (1-4% of total nodule heme) during senescence, indicating that it is not an …

Monoclonal Antibodies Specific For Mercuric Ions, Dwane E. Wylie, Di Lu, Larry D. Carlson, Randy Carlson, K. Funda Babacan, Sheldon M. Schuster, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Monoclonal antibodies (mAbs) that react with soluble mercuric ions have been produced by i 'ection of BALB/c mice with a hapten-carrier complex designed to maximize exposure of the metal to the immune system. Three hybridomas producing antibodies that reacted with bovine serum albumin (BSA)-glutathione-HgCI, but not with BSAglutathione, were isolated from the spleen of a mouse given multiple iqjections with glutathione-HgCl conjugated to keyhole limpet hemocyanin. Stable subclones were established from two of these antibodies, designated mAb 4A10 and mAb IF10. The binding of both antibodies to immobilized BSAglutathione- HgCl was inhibited by soluble HgCl₂, and dissociation constants …

Light Activation Of Maize Phosphoenolpyruvate Carboxylase Protein-Serine Kinase Activity Is Inhibited By Mesophyll And Bundle Sheath-Directed Photosynthesis Inhibitors, Jin-An Jiao, Raymond Chollet

Department of Biochemistry: Faculty Publications

C₄ phosphoenolpyruvate carboxylase (PEPC) is post-translationally regulated by reversible phosphorylation of a specific N-terminal seryl residue in response to light/dark transitions of the parent leaf tissue. The protein-serine kinase (PEPC-PK) that phosphorylates/activates this mesophyll-cytoplasm target enzyme is slowly, but strikingly, activated by high light and inactivated in darkness in vivo by a mechanism involving cytoplasmic protein synthesis/degradation as a primary component. In this report, evidence is presented indicating that the inhibition of Calvin cycle activity by a variety of mesophyll (3-(3,4-dichlorophenyl)-1,1-dimethylurea, isocil, methyl viologen) and bundle sheath (DL-glyceraldehyde)-directed photosynthesis inhibitors blocks the light activation of maize (Zea mays …

Purification And Characterization Of Soybean Root Nodule Ferric Leghemoglobin Reductase, Lin Ji, Stephen Wood, Manuel Becana, Robert V. Klucas

Department of Biochemistry: Faculty Publications

A ferric leghemoglobin reductase from the cytosol of soybean (Glyclne max) root nodules was purified to homogeneity and partlafly characterized. The enzyme is a flavoprotein with flavin adenine dinuclotide as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kilodaltons. The pure enzyme shows a high activity for ferric leghemoglobin reduction with NADH as the reductant in the absence of any exogenous mediators. The enzyme also exhibits NADH-dependent 2,6-dichloroindophenol reductase activity. A sequence of the first 50 N-terminal amino acids of the purified protein was obtained. Comparisons with known protein sequences …

Detection Of Mercuric Ions In Water By Elisa With A Mercury- Specific Antibody, Dwane E. Wylie, Larry D. Carlson, Randy Carlson, Fred W. Wagner, Sheldon M. Schuster

Department of Biochemistry: Faculty Publications

An immunoassay that detects mercuric ions in water at concentrations of 0.5 ppb and above is described. The assay utilizes a monoclonal antibody that binds specifically to mercuric ions immobilized in wells of microtiter plates. Within the range of 0.5-10 ppb mercury, the absorbance in the enzyme-linked immunosorbent assay (ELISA) is linear to the log of the mercuric ion concentration. The quantitation of mercury by ELISA correlates closely with results from cold-vapor atomic absorption. Other divalent metal cations do not interfere with the assay, although there is interference in the presence of 1 mM chloride ions. The optimum pH for …

Photosynthetic Electron Transport In Genetically Altered Photosystem Ii Reaction Centers Of Chloroplasts, Robin A. Roffey, John H. Golebeck, C. Russ Hille, Richard T. Sayre

Department of Biochemistry: Faculty Publications

Using a cotransformation system to identify chloroplast transformants in Chlamydomonas reinhardtii, we converted histidine-195 of the photosystem H reaction center D1 protein to a tyrosine residue. The mutants were characterized by a reduced quantum efficiency for photosynthetic oxygen evolution, which varied in a pH-dependent manner, a reduced capacity to oxidize artificial donors to photosystem II, and P680⁺) reduction kinetics (microsecond) that were essentially similar to wild type. In addition, a dark-stable radical was detected by ESR in mutant photosystem II particles but not in wild-type particles. This radical was similar in g value and lineshape to chlorophyll …

Protein Turnover As A Component In The Light/Dark Regulation Of PhosphoEnolPyruvate Carboxylase Protein-Serine Kinase Activity In C4 Plants, Jin-An Jiao, Cristina Echevarría, Jean Vidal, Raymond Chollet

Department of Biochemistry: Faculty Publications

Maize leaf phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.311 protein-serine kinase (PEPC-PK) phosphorylates serine-15 of its target enzyme, thus leading to an increase in catalytic activity and a concomitant decrease in malate sensitivity of this cytoplasmic C₄ photosynthesis enzyme in the light. We have recently demonstrated that the PEPC-PK activity in maize leaves is slowly, but strikingly, increased in the light and decreased in darkness. In this report, we provide evidence that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of C₄ monocots (maize, sorghum) and dicots (Portulaca oleracea) in the …

Nicotinate, Nicotinamide, And The Reactivity Of Leghemoglobin In Soybean Root Nodules, Robert V. Klucas, Cyril A. Appleby

Department of Biochemistry: Faculty Publications

Nicotinate has been postulated to interfere with the binding of O₂ to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if …

Posttranslational Regulation Of Phosphoenolpyruvate Carboxylase In C4 And Crassulacean Acid Metabolism Plants, Jin-An Jiao, Raymond Chollet

Department of Biochemistry: Faculty Publications

Control of C₄ photosynthesis and Crassulacean acid metabolism (CAM) is, in part, mediated by the diel regulation of phosphoenolpyruvate carboxylase (PEPC) activity. The nature of this regulation of PEPC in the leaf cell cytoplasm of C₄ and CAM plants is both metabolite-related and posttranslational. Specifically, the regulatory properties of the enzyme vary in accord with the physiological activity of C₄ photosynthesis and CAM: PEPC is less sensitive to feedback inhibition by L-malate under light (C₄ plants) or at night (CAM plants) than in darkness (C₄) or during the day (CAM). While the view that …

In Vivo Regulatory Phosphorylation Site In C4-Leaf Phosphoenolpyruvate Carboxylase From Maize And Sorghum, Jin-An Jiao, Jean Vidal, Cristina Echevarría, Raymond Chollet

Department of Biochemistry: Faculty Publications

Reversible seryl-phosphorylation contributes to the light/dark regulation of C₄-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malatesensitivity of the target enzyme has been recently identified as Ser-15 in ³²P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [³²P]phosphopeptides were isolated and purified from in vivo ³²P-labeled maize and …

Primary Sequence Of The Escherichia Coli Fadba Operon, Encoding The Fatty Acid-Oxidizing Multienzyme Complex, Indicates A High Degree Of Homology To Eucaryotic Enzymes, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

In Escherichia coli at least five enzyme activities required for the beta-oxidation of fatty acids are associated with a multienzyme complex composed of two subunits in α₂β₂ conformation (A. Pramanik et al., J. Bacteriol. 137:469-473, 1979). In the present work, the DNA sequence of the genes encoding these two subunits, fadB and fadA, has been determined. The direction of transcription was from fadB to fadA rather than from fadA to fadB, as suggested previously (S. K. Spratt et al., J. Bacteriol. 158:535-542, 1984). Only 10 nucleotides separated the coding sequences for the two peptides, confirming …

Enzymatic And Nonenzymatic Mechanisms For Ferric Leghemoglobin Reduction In Legume Root Nodules, Manuel Becana, Robert V. Klucas

Department of Biochemistry: Faculty Publications

Evidence is presented for the operation in nodules of at least four systems for restoring functional ferrous leghemoglobin (Lb²⁺) from its inactive, ferric form. (i) Reduction of ferric leghemoglobin (Lb³⁺) by a reductase. The enzyme is a flavoprotein of 100 kDa with two equally sized subunits and exhibits a K_m of 9 µM for soybean Lb³⁺ component a and a K. of 51 µM for NADH. NADPH is only 30% (initial velocities) as effective as NADH. Lb³⁺ reductase converts 215 nmol of Lb³+to Lb²⁺ •CO (or Lb²⁺ •O_{2 …}

Role Of Metabolites In The Reversible Light Activation Of Pyruvate,Orthophosphate Dikinase In Zea Mays Mesophyll Cells In Vivo, Chrissi A. Roeske, Raymond Chollet

Department of Biochemistry: Faculty Publications

Whole leaf and mesophyll cell concentrations of pyruvate, phosphoenolpyruvate (PEP), ATP, and ADP were determined in Zea mays during the reversible light activation of pyruvate, orthophosphate dikinase in vivo. Mesophyll cell levels of the four metabolites were estimated by extrapolation from values in freeze-quenched leaf samples that were fractionated by differential filtration through nylon mesh nets (adapted from M Stitt, HW Heldt [1985] Planta 164: 179-188). During the 3 minutes required for complete light activation of dikinase, pyruvate levels in the mesophyll cell decreased (from 166 ± 15 to 64 ± 10 nanomoles per milligram of chlorophyll [nmol/mg Chi]) …

Chloroplast Thylakoid Protein Phosphatase Is A Membrane Surface-Associated Activity, Gongqin Sun, Doreen Bailey, Michael W. Jones, John Markwell

Department of Biochemistry: Faculty Publications

Chloroplast thylakoid protein phosphatase activity was measured using ³²P-labeled histone as an exogenous substrate and an assay of the ³²P released involving formation of a phosphomolybdate complex and organic extraction. The activity was liberated from wheat (Triticum aestivum) thylakoids by washing the membranes in NaCI-containing solutions followed by centrifugation. The liberated phosphatase activity had a pH optimum of approximately 6.75, was inhibited by addition of 10 millimolar EDTA or EGTA, and was stimulated by addition of millimolar amounts of dithiothreitol, magnesium, manganese, or calcium ions. The rate of thylakoid protein dephosphorylation was decreased following liberation of a portion …

Multiple Isotope Effects On Enzyme-Catalyzed Reactions, Marion H. O'Leary

Department of Biochemistry: Faculty Publications

Isotope effects have long been popular as a method for studying mechanisms of chemical reactions (1, 2). Application of this method to enzymatic reac­tions has come slowly, in part because of the difficulty of making measurements of the necessary precision, and in part because of the difficulty of interpreting the variations in rate that occur in multistep reactions. The theory and practice of isotope effects has now reached the stage where a variety of interesting mechanistic studies are possible (3-9), including use of heavy­ atom isotope effects (5), application to multireactant enzymes (9a, 10), pH dependence of isotope effects (11, …

Nucleotide Sequence Of The Fadr Gene, A Multifunctional Regulator Of Fatty Acid Metabolism In Escherichia Coli, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

The Escherichia coli fadR gene is a multifunctional regulator of fatty acid

and acetate metabolism. In the present work the nucleotide sequence of the

1.3 kb DNA fragment which encodes FadR has been determined. The coding

sequence of the fadR gene is 714 nucleotides long and is preceded by a typical

E. coli ribosome binding site and is followed by a sequence predicted to be

sufficient for factor-independent chain termination. Primer extension experiments

demonstrated that the transcription of the fadR gene initiates with an

adenine nucleotide 33 nucleotides upstream from the predicted start of

translation. The derived fadR peptide has …

Reduced Co2/O2 Specificity Of Ribulose-Bisphosphate Carboxylase/Oxygenase In A Temperature-Sensitive Chloroplast Mutant Of Chlamydomonas, Zhixiang Chen, Chris J. Chastain, Souhail R. Al-Abed, Raymond Chollet, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25°C but fails to grow photosynthetically at 35°C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25°C, but less than 15% when grown at 35°C. Pulse-labeling with ³⁵S showed that the decrease in enzyme level at the restrictive temperature was not a result of reduced synthesis of enzyme subunits. The CO₂/O₂ specificity factor (V_cK_o/V_oK_c, where V_c and V_o are V_{max …}