Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

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 …

The Binding Of Host-Selective Toxin Analogs To Mitochondria From Normal And 'Texas' Male Sterile Cytoplasm Maize, Kurt A. Frantzen, J. M. Daly, Herman W. Knoche

Department of Biochemistry: Faculty Publications

Tritium-labeled toxin analogs were prepared by reduction with NaB³H₄ of either the toxin from Helminthosporium maydis race T or a toxin component from Phyllosticta maydis. These reduced analogs had high radiochemical specific activities, high biological activities, and plant specificities identical to the native toxins. A filtration assay was developed to test the binding of these labeled analogs to isolated- mitochondria. Binding was not energy dependent nor was there measurable matrical uptake. The analogs were shown to be lipophilic, a characteristic which gave rise to considerable nondisplaceable binding. Under conditions limiting nondisplaceable binding, -the displaceable binding was …

In Vitro Phosphorylation Of Maize Leaf Phosphoenolpyruvate Carboxylase, Raymond J. A. Budde, Raymond Chollet

Department of Biochemistry: Faculty Publications

Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with ³²P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ -32P]ATP or [β-³²P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acidstable 94 …

Cloning And Characterization Of A Gene (Fadr) Involved In Regulation Of Fatty Acid Metabolism In Escherichia Coli, Concetta C. Dirusso, William D. Nunn

Department of Biochemistry: Faculty Publications

The regulatory gene fadR has been previously characterized by classical genetic means as a diffusible protein which exerts negative control over fatty acid degradation and acetate metabolism. fadR has also been implicated in the regulation of unsaturated fatty acid biosynthesis. To facilitate the identification of the product of the fadR gene and to study the mechanism by which this multifunctional regulatory gene exerts its control, we cloned a segment of DNA containing the fadR gene in the phage vector λL47. Subsequent subcloning of a segment of the chromosomal DNA from the λfadR⁺ phage into various plasmid vectors resulted in …

Reduction Of Ferric Leghemoglobin In Soybean Root Nodules, Keuk-Ki Lee, Robert V. Klucas

Department of Biochemistry: Faculty Publications

Reduction of ferric leghemoglobin to ferrous leghemoglobin in soybean nodules (Glycine max [L.] Meff. cv Woodworth) was studied using a spectrophotometer equipped with an in-cell space diffuse reflectance accessory. Nodule slices prepared and scanned under nitrogen gas showed a ferrous leghemoglobin absorption spectrum. Nodule slices equilibrated with 100% O₂ or air exhibited two absorption bands characteristic of oxygenated leghemoglobin. The addition of CO shifted those bands to CO leghemoglobin absorption bands. Potassium ferricyanide was not effective in oxidizing ferrous to feffic leghemoglobin in nodule slices. However, ferric leghemoglobin was formed by treating the nodule slices with hydroxylamine, and …

The Role Of Recognition In Plant Disease, J. M. Daly

Department of Biochemistry: Faculty Publications

As noted by Sequeira (110,111), the term recognition has become very popular in the literature of host-pathogen interactions over the last ten years. Despite recent facile usage, its meaning is not always clear to me. Recognition is defined by Clarke & Knox (16) as the "initial event in cell-cell communication that elicits a defined biochemical, physiological, or morphological response." This requires the not-so-easy task of defining "initial event" and "communica­ tion" with precise chemical or physical mechanisms. For pathologists, Se­ queira (110) has provided a more restricted meaning: "An early specific event that triggers a rapid, overt response by the …

Reversible Dark-Induced Senescence Of Soybean Root Nodules, N. E. Pfeiffer, Nasir S.A. Malik, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Nodule senescence was induced in intact soybean Glycine max. (L.) Merr., cv Woodworth plants by an 8-day dark treatment. Dark-induced senescence resulted in the complete loss of acetylene reduction activity, a 67% loss of total soluble protein, and an almost complete loss in total leghemoglobin of nodule extracts. Isoelectric focusing gels demonstrated a preferential loss of certain proteins, which was correlated with an increase in endoprotease specific activity toward azocasein. Nodules were completely green after the 8-day dark treatment. If plants were returned to a normal photoperiod after 8 days in the dark, nodules recovered from the dark treatment in …

Proteolytic Activity In Soybean Root Nodules: Activity In Host Cell Cytosol And Bacteroids Throughout Physiological Development And Senescence, Nancy E. Pfeiffer, Cecilia M. Torres, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merril cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in …

Peptidohydrolases Of Soybean Root Nodules, Nasir S. A. Malik, N. E. Pfeiffer, D. R. Williams, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminoptidase, and carboxypeptidase actiities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-LLeu-β-naphthylamide, while the second hydrolyzed n-benzoyl-L-Arg-/8- naphthylamine (Bz-L-Arg-/βNA), n-benzoyl-L-Arg-β-nitroanilide (Bz-L-Arg-βNA), and azocasein. The third endopeptidase hydrolyzed Bz-L-Arg-βNA, Bz-L-Arg-βNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. AU of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.

Recollections, George W. Beadle

Department of Biochemistry: Faculty Publications

In these exciting times when elementary and high schools teach modern biology, including many of the intricacies of biochemical genetics, the long slow process by which our present knowledge in this area was gained is not often fully appreciated. A third of a century elapsed before Mendel’s work was "rediscovered" and properly appreciated. Archibald E. Garrod’s (1) prophetic appreciation of the relation genetics and biochemistry, beginning soon after the so-called rediscovery of Mendel, lay fallow for more than forty years despite the fact that he published widely and relatively voluminously. As late as a quarter of a century after the …

Characterization Of A Fatty Acid Synthetase From Corynebacterium Diphtheriae, Herman W. Knoche, Kirston E. Koths

Department of Biochemistry: Faculty Publications

A fatty acid synthetase from Corynebacterium diphtheriae has been purified to a specific activity of 450 nmoles of malonyl coenzyme A incorporated per min per mg. The enzyme is optimally active in 0.5 M phosphate buffer. C. diphtheriae appears to be the most primitive organism having a multienzyme complex for fatty acid synthesis.

Palmityl Coenzyme A Inhibition Of Fatty Acid Synthesis, Herman Knoche, Theodore W. Esders, Kirston Koths, Konrad Bloch

Department of Biochemistry: Faculty Publications

The effects of acyl-CoA derivatives (C₈ to C₂₀) on the activity

of the fatty acid synthetases from yeast and Corynebacterium

diphtheriae have been examined. Both enzyme

systems are inhibited by the longer chain acyl thioesters

(C₁₆ to C₂₀) and protected against this inhibition by bovine

serum albumin (BSA). Identical relief from acyl-CoA inhibition

is provided by the 6-0-methylglucose-containing

lipopolysaccharide (MGLP), from Mycobacterium phlei. It

is shown that MGLP forms a stable complex with palmitylCoA.

This interaction accounts for the BSA-like effects of

the polysaccharide. BSA and MGLP have two further effects

on the fatty …

The Structure Of An Ornithine-Containing Lipid From Thiobacillus Thiooxidans, Herman W. Knoche, Jessup M. Shively

Department of Biochemistry: Faculty Publications

The structure of an ornithine-containing lipid from Thio-bacillus thiooxidans

has been elucidated. Methanolysis of

the lipid released methyl cis-ll,lZ-methylene-Z-hydroxyoctadecanoate.

Acid hydrolysis of the residue yielded ornithine

and a mixture of fatty acids, the major components of

which were 3-hydroxyhexadecanoic and 2-hexadecenoic

acids. Identification of the 3-hydroxy fatty acid was based

on the thin layer chromatographic mobilities of the acid, its

methyl ester, the methyl ether, and acetate derivatives of its

methyl ester, on the equivalent chain lengths of the derivatives

of the acid and the acid obtained by oxidation of the

natural acid with permanganate, and on mass spectral studies. …

The Identification Of Cis-Ii, I2-Methylene-2-Hydroxyoctadecanoic Acid From Thiobacillus Thiooxidans, H. W. Knoche, J. M. Shively

Department of Biochemistry: Faculty Publications

A polar fatty acid has been observed as a component of an

ornithine-containing lipid of Thiobacillus thiooxidans. A

comparison of thin layer chromatographic mobilities of reference

compounds to those of the natural acid and its derivatives

suggested that the acid was a 2-hydroxy fatty acid.

The presence of a cyclopropane function in the acid was

indicated by l4e-Iabeling experiments and infrared spectroscopy.

Mass spectrometry of the methyl ester and the acetylated

methyl ester of the natural acid provided a molecular

weight for the acid. Equivalent chain lengths were determined

for the natural acid, the acid obtained by oxidative

decarboxylation of …

Carbohydrate And Lipid Metabolism During Germination Of Uredospores Of Puccinia Graminis Tritici, J. M. Daly, H. W. Knoche, M. V. Wiese

Department of Biochemistry: Faculty Publications

Uredospores of Puccinia gramintis (Pers.) tritici (Eriks. and Henn.) were uniformly labeled with ¹⁴C by permitting the host (Triticuml aestivum L.) to carry out photosynthesis in ¹⁴CO₂ during the process of spore production by the obligate parasite. The use of ¹⁴C labeled spores provided advantages in a study of the utilization of endogenous substrates at frequent intervals with small amounts of spores under conditions conducive to germination.

Concerning The Site Of Nitrogen Absorption In Rats Fed Autoclaved Or Raw Soybean Oil Mea, Raymond Borchers

Department of Biochemistry: Faculty Publications

Carroll, Hensley, and Graham (1) have concluded that much of the nitrogen absorption in rats fed raw soybean oil meal must take place in the cecum. This conclusion was reached from data showing that the apparent digestibility of raw soybean nitrogen in the terminal 20% of the small intestine was 32.65%, whereas in the feces the value was 76.96%. Values reported, for apparent digestibility of heated soybean nitrogen were 78.66% and 81.78%, respectively. This observation presented a notable advance in explaining the lower nutritive value of raw soybeans compared with autoclaved soybeans. It therefore seemed advisable to repeat this work …

Riboflavin Production By Molds, George L. Peltier, Raymond Borchers

Department of Biochemistry: Faculty Publications

Very little information is at hand regarding the ability of molds to synthesize riboflavin. Few citations pertaining directly to riboflavin production by true

molds are available. Pontovich (1943) found as much as 2 mg riboflavin per g

of Aspergillus flavus mycelium. Tanner et at. (1945) determined the quantity of

riboflavin in the submerged fermentation media of Penicillium chrysogenum.

The highest value found was 1. 36 mg per ml. The primary purpose of this study

was to screen several hundred isolates, recently obtained from soil, crop residues,

and composts, for their ability to produce riboflavin on a wheat bran substrate.

Threonine Deficiency In Hydrolysates Of Zein Prepared By Autoclaving, Raymond Borchers, John R. Totter, Clarence P. Berg

Department of Biochemistry: Faculty Publications

In a previous paper (Borchers and Berg, 1942) we showed that

autoclaving zein with sulfuric acid longer than necessary for complete

hydrolysis causes destruction and racemization; either of

these might account for the failure of such a hydrolysate to

promote growth in young rats when substituted for a hvdrolysate,

prepared by refiuxing, in a diet which produced moderate growth.

It seemed to us that this deterioration in the dietary protein might

well be the result primarily of essential amino acid deficiencies

which could be detected and overcome by appropriate supplementation.

Because threonine is known to be present in zein in …