Biochemistry Commons^™

Interactions Between Small Heat Shock Protein Subunits And Substrate In Small Heat Shock Protein-Substrate Complexes, Kenneth L. Friedrich,, Kim C. Giese, Nicole R. Buan, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

Small heat shock proteins (sHSPs) are dynamic oligomeric proteins that bind unfolding proteins and protect them from irreversible aggregation. This binding results in the formation of sHSP-substrate complexes from which substrate can later be refolded. Interactions between sHSP and substrate in sHSP-substrate complexes and the mechanism by which substrate is transferred to ATP-dependent chaperones for refolding are poorly defined. We have established C-terminal affinity-tagged sHSPs from a eukaryote (pea HSP18.1) and a prokaryote (Synechocystis HSP16.6) as tools to investigate these issues. We demonstrate that sHSP subunit exchange for HSP18.1 and HSP16.6 is temperature-dependent and rapid at the optimal growth temperature …

Real-Time Study Of Multidrug Resistance Mechanism In Pseudomonas Aeruginosa Using Nanoparticle Optics And Single Live Cell Imaging, Sophia Vasou Kyriacou

Chemistry & Biochemistry Theses & Dissertations

This thesis centers on the study of the xenobiotic efflux system in Pseudomonas aeruginosa, which is a ubiquitous bacterium. It resists many structurally and functionally diverse substrates due to expression of Mex-extrusion pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM systems. Despite extensive research, the structure and mechanism of multidrug resistance is unclear (1-9). For example, (i) how do MexA, MexB and OprM proteins assemble to extrude antibiotics? (ii) What is the antibiotic susceptibility of MexA, MexB, and OprM proteins? (iii) How do substrates cross the outer membrane of P. aeruginosa? (iv) Where are antibiotics accumulated inside the cell? This thesis …

Transmembrane Movement Of Exogenous Long-Chain Fatty Acids: Proteins, Enzymes, And Vectorial Esterification, Paul N. Black, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

INTRODUCTION......................................................................................................................................................454

FATTY ACIDS AND BIOLOGICAL MEMBRANES.............................................................................................455

Fatty Acid Transport Defined ...............................................................................................................................456

MODEL SYSTEMS TO INVESTIGATE FATTY ACID TRANSPORT ..............................................................456

Genetic Foundations of Fatty Acid Transport....................................................................................................456

PROTEINS IMPLICATED IN FATTY ACID TRANSPORT ...............................................................................456

Fatty Acid Translocase..........................................................................................................................................457

Fatty Acid Binding Protein—Membrane Bound ................................................................................................457

Fatty Acid Transport Protein...............................................................................................................................458

The Long-Chain Fatty Acid Transport Protein FadL .......................................................................................458

Fatty Acyl-CoA Synthetase ...................................................................................................................................459

VECTORIAL ACYLATION: ONE MECHANISM OPERATIONAL IN FATTY ACID TRANSPORT...........460

THE BACTERIAL PARADIGM ...............................................................................................................................460

Energetics of Fatty Acid Transport in Gram-Negative Bacteria......................................................................460

The Fatty Acid Transporter FadL........................................................................................................................461

The Fatty Acyl-CoA Synthetase FadD..................................................................................................................461

Structural Considerations of FACS .....................................................................................................................462

FATTY ACID TRANSPORT AND ACTIVATION IN YEAST: …

The Many Faces Of Vitamin B12: Catalysis By Cobalamin-Dependent Enzymes, Ruma Banerjee, Stephen W. Ragsdale

Department of Biochemistry: Faculty Publications

Vitamin B₁₂ is a complex organometallic cofactor associated with three subfamilies of enzymes: the adenosylcobalamin-dependent isomerases, the methylcobalamin-dependent methyltransferases, and the dehalogenases. Different chemical aspects of the cofactor are exploited during catalysis by the isomerases and the methyltransferases. Thus, the cobalt-carbon bond ruptures homolytically in the isomerases, whereas it is cleaved heterolytically in the methyltransferases. The reaction mechanism of the dehalogenases, the most recently discovered class of B₁₂ enzymes, is poorly understood. Over the past decade our understanding of the reaction mechanisms of B₁₂ enzymes has been greatly enhanced by the availability of large amounts of enzyme …

Electrically Mediated Delivery Of Vector Plasmid Dna Elicits An Antitumor Effect, L. Heller, D. Coppola

Bioelectrics Publications

In vivo electroporation is an efficient means of increasing plasmid DNA delivery to normal tissues, such as skin and muscle, as well as directly to tumors. In the experiments described here, plasmid DNA was delivered by in vivo electroporation to B16 mouse melanomas using two very different pulsing protocols. Reporter expression increased 21- or 42-fold, respectively with electroporation over injection alone. The growth of experimental melanomas with an approximate diameter of 4 mm on the day of treatment was monitored after electroporation delivery of reporter plasmid DNA. Remarkably, short-term complete regressions using one of these pulsing protocols occurred in up …

The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Of Salmonella Enterica Requires The 2'-Oh Group Of Atp For Function And Yields Inorganic Triphosphate As Its Reaction Byproduct, Maris V. Fonseca, Nicole R. Buan, Alexander R. Horswill, Ivan Rayment, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The specificity of the ATP:corrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica serovar Typhimurium LT2 for its nucleotide substrate was tested using ATP analogs and alternative nucleotide donors. The enzyme showed broad specificity for the nucleotide base and required the 2’-OH group of the ribosyl moiety of ATP for activity. ³¹P NMR spectroscopy was used to identify inorganic triphosphate (PPP_i) as the byproduct of the reaction catalyzed by the CobA enzyme. Cleavage of triphosphate into pyrophosphate and orthophosphate did not occur, indicating that triphosphate cleavage was not required for release of the adenosylcorrinoid product. Triphosphate was a strong …

Vectorial Acylation In Saccharomyces Cerevisiae, Zhiying Zou, Fumin Tong, Nils J. Færgeman, Claus Børsting, Paul N. Black, Concetta Dirusso

Department of Biochemistry: Faculty Publications

In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions between these proteins. Multicopy extragenic suppressors were selected in strains carrying deletions in FAA1 and FAA4 or FAA1 and FAT1. Each strain is unable to grow under synthetic lethal conditions when exogenous long-chain fatty acids are required, and neither strain accumulates the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ indicating a fatty acid transport defect. By using these …

The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Of Salmonella Enterica Requires The 2’-Oh Group Of Atp For Function And Yields Inorganic Triphosphate As Its Reaction Byproduct, Maris V. Fonseca, Nicole R. Baun, Alexander R. Horswill, Ivan Rayment, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The specificity of the ATP:corrinoid adenosyltransferase

(CobA) enzyme of Salmonella enterica serovar

Typhimurium LT2 for its nucleotide substrate was

tested using ATP analogs and alternative nucleotide donors.

The enzyme showed broad specificity for the nucleotide

base and required the 2’-OH group of the ribosyl

moiety of ATP for activity. ³¹P NMR spectroscopy

was used to identify inorganic triphosphate (PPP_i) as

the byproduct of the reaction catalyzed by the CobA

enzyme. Cleavage of triphosphate into pyrophosphate

and orthophosphate did not occur, indicating that

triphosphate cleavage was not required for release of

the adenosylcorrinoid product. Triphosphate was …

Electrically Mediated Plasmid Dna Delivery To Hepatocellular Carcinomas In Vivo, L. Heller, M. J. Jaroszeski, D. Coppola, C. Pottinger, R. Gilbert, Richard Heller

Bioelectrics Publications

Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.

Multiple Factors Independently Regulate Hila And Invasion Gene Expression In Salmonella Enterica Serovar Typhimurium, Robin L. Lucas, C. Phoebe Lostroh, Concetta C. Dirusso, Michael P. Spector, Barry L. Wanner, Catherine A. Lee

Department of Biochemistry: Faculty Publications

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more

about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In

addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the

pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence

of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the

PhoR-PhoB two-component regulatory system, suggesting that hilA expression may …

Multiple Factors Independently Regulate Hila And Invasion Gene Expression In Salmonella Enterica Serovar Typhimurium, Robin L. Lucas, C. Phoebe Lostroh, Concetta C. Dirusso, Michael P. Spector, Barry L. Wanner, Catherine A. Lee

Department of Biochemistry: Faculty Publications

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more

about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In

addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the

pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence

of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the

PhoR-PhoB two-component regulatory system, suggesting that hilA expression may …

Two-Step Processing Of Human Frataxin By Mitochondrial Processing Peptidase, Patrizia Cavadini, Jiri Adamec, Franco Taroni, Oleksandr Gakh, Grazia Isaya

Department of Biochemistry: Faculty Publications

We showed previously that maturation of the human

frataxin precursor (p-fxn) involves two cleavages by the

mitochondrial processing peptidase (MPP). This observation

was not confirmed by another group, however,

who reported only one cleavage. Here, we demonstrate

conclusively that MPP cleaves p-fxn in two sequential

steps, yielding a 18,826-Da intermediate (i-fxn) and a

17,255-Da mature (m-fxn) form, the latter corresponding

to endogenous frataxin in human tissues. The two cleavages

occur between residues 41–42 and 55–56, and both

match the MPP consensus sequence RX ↓ (X/S). Recombinant

rat and yeast MPP catalyze the pài step 4 and 40 …

Urea Hydrolysis Using Immobilized Urease, Anita Arullan

Student Works (2000-2009)

An inexpensive carrier matrix. vermiculite was used for immobilizing urease. A cost effective and simple method of coupling was developed using vermiculite. Immobilization of urease on Amberlite MB-land Nylon-6 tube were also studied. Activities of free and immobilized urease were studied and compared: Among all methods used for immobilization of urease. the method of coupling the enzyme to vermiculite gave the highest retained activity of 82% (Method I) and 89% (Method 2) at pH 6.0 and 5.5 respectively. Urease immobilized by Method I retained 69% and 30% of its original activity and Method 2 (using glutaraldehyde as coupling agent) retained …

Yeast And Human Frataxin Are Processed To Mature Form In Two Sequential Steps By The Mitochondrial Processing Peptidase, Steven S. Branda, Patrizia Cavadini, Jiri Adamec, Frantisek Kalousek, Franco Taroni, Grazia Isaya

Department of Biochemistry: Faculty Publications

Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich’s ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not …

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 …

Pattern Of Disease After Murine Hepatitis Virus Strain 3 Infection Correlates With Macrophage Activation And Not Viral Replication, M. Pope, O. Rotstein, E. Cole, S. Sinclair, Rebecca D. Parr, B. Cruz, R. Fingerote, S. Chung, R. Gorczynski, L. Fung, J. Leibowitz, Y. S. Rao, G. Levy

Faculty Publications

Murine hepatitis virus strain (MHV-3) produces a strain-dependent pattern of disease which has been used as a model for fulminant viral hepatitis. This study was undertaken to examine whether there was a correlation between macrophage activation and susceptibility or resistance to MHV-3 infection. Peritoneal macrophages were isolated from resistant A/J and susceptible BALB/cJ mice and, following stimulation with MHV-3 or lipopolysaccharide (LPS), analyzed for transcription of mRNA and production of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), mouse fibrinogen-like protein (musfiblp), tissue factor (TF), leukotriene B4, and prostaglandin E2 (PGE2). Macrophages from BALB/cJ mice produced …

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 …

Molecular Cloning And Rare Cleavage Mapping Of Human 2p, 6q, 8q, 12q, And 18q Telomeres, Roberto A. Macina, Ken Morii, Xue-Lan Hu, Dimitri G. Negorev, Chrysanthe Spais, Lisa A. Ruthig, Harold C. Riethman

School of Medical Diagnostics & Translational Sciences Publications

Large terminal fragments of human chromosomes 2p, 6q, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from 11 unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned Fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.

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 …

Sds Non-Acrylamide Polymeric Gel-Filled Capillary Electrophoresis For Molecular Size Separation Of Protein, Devon Andres

Honors Theses

Sodium dodecyl sulfide (SDS) non-acrylamide gel-filled capillary columns are a new technology being used for analysis and separation of biotechnology-derived proteins. This research was to compare this new technology to the current methods of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The molecular mass of four different recombinant proteins were determined by two commercialized SDS non-acrylamide gel-filled capillary columns, SDS-PAGE, and HPSEC. The data obtained showed that the SDS non-acrylamide gel-filled capillary columns were compatible with the SDS-PAGE technique for molecular mass determination. HPSEC was shown to be unreliable for molecular weight determination. SDS non-acrylamide gel-filled capillary …

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 …

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 …

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 …

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 …