Biotechnology Commons^™

High-Throughput Screening For Fatty Acid Uptake Inhibitors In Humanized Yeast Identifies Atypical Antipsychotic Drugs That Cause Dyslipidemias, Hong Li, Paul N. Black, Aalap Chokshi, Angel Sandoval-Alvarez, Ravi Vatsyayan, Whitney Sealls, Concetta Dirusso

Department of Biochemistry: Faculty Publications

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12). The library used consisted of 2,080 compounds with known biological activities. Of these, ~1.8% reduced cell-associated C1-BODIPY-C12 fluorescence and were selected as potential inhibitors of human FATP2- mediated fatty acid …

A New Strategy For Assessing Selenoprotein Function: Sirna Knockdown/Knock-In Targeting The 3'-Utr, Min-Hyuk Yoo, Xue-Ming Xu, Anton A. Turanov, Bradley A. Carlson, Vadim N. Gladyshev, Dolph L. Hatfield

Department of Biochemistry: Faculty Publications

Selenocysteine insertion into protein in mammalian cells requires RNA elements in the 3'-untranslated regions (3'-UTRs) of selenoprotein genes. The occurrence of these conserved sequences should make selenoproteins particularly amenable for knockdown/knock-in strategies to examine selenoprotein functions. Herein, we utilized the 3'-UTR of various selenoproteins to knock down their expression using siRNAs and then knock in expression using constructs containing mutations within the target region. Thioredoxin reductase 1 (TR1) knockdown in a mouse kidney cell line resulted in the cells growing about 10% more slowly, being more sensitive to UV radiation, and having increased apoptosis in response to UV than control …

Mechanistic Studies Of The Long Chain Acyl-Coa Synthetase Faa1p From Saccharomyces Cerevisiae, Hong Li, Elaina M. Melton, Steven Quackenbush, Concetta C. Dirusso, Paul N. Black

Department of Biochemistry: Faculty Publications

Long chain acyl-CoA synthetase (ACSL; fatty acid CoA ligase: AMP forming; EC 6.2.1.3) catalyzes the formation of acyl-CoA through a process, which requires fatty acid, ATP and coenzymeA as substrates. In the yeast Saccharomyces cerevisiae the principal ACSL is Faa1p (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faa1p is a principal component of a fatty acid transport/activation complex that also includes the fatty acid transport protein Fat1p. In the present work hexameric histidine tagged Faa1p was purified to homogeneity through a two-step process in the …

Evidence For A Pro-Oxidant Intermediate In The Assembly Of Cytochrome Oxidase, Oleh Khalimonchuk, Amanda Bird, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The hydrogen peroxide sensitivity of cells lacking two proteins,

Sco1 and Cox11, important in the assembly of cytochrome

c oxidase (CcO), is shown to arise from the transient accumulation

of a pro-oxidant heme A-Cox1 stalled intermediate. The

peroxide sensitivity of these cells is abrogated by a reduction in

either Cox1 expression or hemeAformation but exacerbated by

either enhanced Cox1 expression or heme A production arising

from overexpression of COX15. Sco1 and Cox11 are implicated

in the formation of the Cu_A and Cu_B sites of CcO, respectively.

The respective wild-type genes suppress the peroxide sensitivities

of sco1∆ …

Coa1 Links The Mss51 Post-Translational Function To Cox1 Cofactor Insertion In Cytochrome C Oxidase Assembly, Fabien Pierrel, Megan L. Bestwick, Paul A. Cobine, Oleh Khalimonchuk, Julia A. Cricco, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is shown to be dependent on a new assembly factor designated Coa1 that associates with the mitochondrial inner membrane. Translation of the mitochondrial-encoded subunits of CcO occurs normally in coa1Δ cells, but these subunits fail to accumulate. The respiratory defect in coa1Δ cells is suppressed by high-copy MSS51, MDJ1 and COX10. Mss51 functions in Cox1 translation and elongation, whereas Cox10 participates in the biosynthesis of heme a, a key cofactor of CcO. Respiration in coa1Δ and shy1Δ cells is enhanced when Mss51 and …

Sequential Processing Of A Mitochondrial Tandem Protein: Insights Into Protein Import In Schizosaccharomyces Pombe, Oleh Khalimonchuk, Martin Ott, Soledad Funes, Kai Ostermann, Gerhard Rödel, Johannes M. Herrmann

Department of Biochemistry: Faculty Publications

The sequencing of the genome of Schizosaccharomyces pombe revealed the presence of a number of genes encoding tandem proteins, some of which are mitochondrial components. One of these proteins (pre-Rsm22- Cox11) consists of a fusion of Rsm22, a component of the mitochondrial ribosome, and Cox11, a factor required for copper insertion into cytochrome oxidase. Since in Saccharomyces cerevisiae, Cox11 is physically attached to the mitochondrial ribosome, it was suggested that the tandem organization of Rsm22-Cox11 is used to covalently tie the mitochondrial ribosome to Cox11 in S. pombe. We report here that pre-Rsm22-Cox11 is matured in two subsequent …

Purification And Initial Biochemical Characterization Of Atp:Cob(I)Alamin Adenosyltransferase (Eutt) Enzyme Of Salmonella Enterica, Nicole R. Baun, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella

enterica was overproduced and enriched to ~70% homogeneity,

and its basic kinetic parameters were determined. Abundant

amounts of EutT protein were produced, but all of it remained

insoluble. Soluble active EutT protein (~70% homogeneous) was

obtained after treatment with detergent. Under conditions in which

cobalamin (Cbl) was saturating, _Km(ATP) = 10 µM, k_cat = 0.03 s^-1,

and V_max = 54.5 nM min^-1. Similarly, under conditions in which

MgATPwas saturating,K_m(Cbl) = 4.1µM, k_cat = 0.06 s^-1, andV_max= …

Computer-Assisted Docking Of Flavodoxin With The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Reveals Residues Critical For Protein-Protein Interactions But Not For Catalysis*, Nicole R. Buan, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobAand FldA interact, a computer model of aCobA∙FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA …

Computer-Assisted Docking Of Flavodoxin With The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Reveals Residues Critical For Protein-Protein Interactions But Not For Catalysis, Nicole R. Baun, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase

(CobA) enzyme of Salmonella enterica sv. Typhimurium

is required to adenosylate de novo biosynthetic intermediates

of adenosylcobalamin and to salvage incomplete and complete

corrinoids from the environment of this bacterium. In vitro,

reduced flavodoxin (FldA) provides an electron to generate the

co(I)rrinoid substrate in the CobA active site. To understand how

CobAand FldA interact, a computer model of aCobA-FldA complex

was generated. This model was used to guide the introduction of

mutations into CobA using site-directed mutagenesis and the synthesis

of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of

CobA …

The Identity Of Proteins Associated With A Small Heat Shock Protein During Heat Stress In Vivo Indicates That These Chaperones Protect A Wide Range Of Cellular Functions, Eman Basha, Garrett J. Lee, Linda A. Breci, Andrew C. Hausrath, Nicole R. Buan, Kim C. Giese, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones and are heat-labile. Thirteen …

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

The Identity Of Proteins Associated With A Small Heat Shock Protein During Heat Stress In Vivo Indicates That These Chaperones Protect A Wide Range Of Cellular Functions, Eman Basha, Garrett J. Lee, Linda A. Breci, Andrew C. Hausrath, Nicole R. Baun, Kim C. Giese, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

The small heat shock proteins (sHSPs) are a ubiquitous

class of ATP-independent chaperones believed to

prevent irreversible protein aggregation and to facilitate

subsequent protein renaturation in cooperation

with ATP-dependent chaperones. Although sHSP chaperone

activity has been studied extensively in vitro, understanding

the mechanism of sHSP function requires

identification of proteins that are sHSP substrates in

vivo. We have used both immunoprecipitation and affinity

chromatography to recover 42 proteins that specifically

interact with Synechocystis Hsp16.6 in vivo during

heat treatment. These proteins can all be released from

Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones

and are heat-labile. …

5-Hydroxydecanoate Is Metabolised In Mitochondria And Creates A Rate-Limiting Bottleneck For Β-Oxidation Of Fatty Acids, Peter J. Hanley, Stefan Dröse, Ulrich Brandt, Rachel A. Lareau, Abir L. Banerjee, D. K. Srivastava, Leonard J. Banaszak, Joseph J. Barycki, Paul P. Van Veldhoven, Jürgen Daut

Department of Biochemistry: Faculty Publications

5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and

has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K+ (KATP)

channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA

(5-HD-CoA), which is a substrate for the first step of β-oxidation. We have now

analysed the complete β-oxidation of 5-HD-CoA using specially synthesised (and purified)

substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared

it with the metabolism of the physiological substrate decanoyl-CoA. At the second step of

β-oxidation, catalysed by enoyl-CoA hydratase, enzyme kinetics were …

Mobilization Of Intracellular Copper Stores By The Ctr2 Vacuolar Copper Transporter, Erin M. Rees, Jaekwon Lee, Dennis J. Thiele

Department of Biochemistry: Faculty Publications

Copper plays an essential role in processes including signaling to the transcription and protein trafficking machinery, oxidative phosphorylation, iron mobilization, neuropeptide maturation, and normal development. Whereas much is known about intracellular mobilization of ions such as calcium, little information is available on how eukaryotic cells mobilize intracellular copper stores. We describe a mechanism by which the Saccharomyces cerevisiae Ctr2 protein provides bioavailable copper via mobilization of intracellular copper stores. Whereas Ctr2 exhibits structural similarity to the Ctr1 plasma membrane copper importer, microscopic and biochemical fractionation studies localize Ctr2 to the vacuole membrane. We demonstrate that Ctr2 mobilizes vacuolar copper stores …

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 …

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 …

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 …

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 …

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 …

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 …

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 …