Purification and characterization of lipases from wheat germ

A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivum) is described. Electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 x x 10(-3) mumol/min per mg protein) was obtained after purification in 61 times. The molecular weight of the enzyme, determined by gel chromatography, was 143 +/- 2 kDa. The optimal conditions for the enzyme were 37 degrees and pH 8.0. Homogeneous preparation of the lipase exhibited high thermal stability: over 20% of original activity was retained after incubation of the preparation at high temperatures (60-90 degrees) for 1 h at pH 8.0.     

Purification and characterization of a lysozome from wheat germ

Several proteins of wheat germ were able to lyse Micrococcus luteus cells. One lysozyme, named W1A, was purified by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and preparative polyacrylamide gel electrophoresis (PAGE) under native conditions. The enzyme had a molecular weight of 25 400 as determined by sodium dodecyl sulfate (SDS)-PAGE. The reducing groups released from the lysis of Micrococcus cell walls by W1A lysozyme were $\text{N-}\text{acetylmuramic}$ acid residues as for hen egg white lysozyme (HEWL). Chitin substrates were hydrolyzed to some extent by this enzyme. With Micrococcus cells as substrate, the pH optimum for W1A lysozyme was 6.0 at an optimal ionic strength of 0.05. Under these conditions, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 166 mg/l with purified Micrococcus cell walls and the V_max value was 0.56 A₅₄₀ unit/min at 22°C. W1A lysozyme exhibited the highest lytic activity at 60°C whereas the enzyme was inactive above 90°C. W1A lysozyme was strongly inhibited by poly-l-lysine and glycol chitosan. This is the first report of the presence of multiple electrophoretic forms of plant lysozyme activity as determined by native PAGE.     

Purification and characterization of wheat germ DNA topoisomerase I (nicking-closing enzyme)

Wheat germ contains an enzyme capable of removing supercoils from circular DNA. We have purified this enzyme using Polymin P fractionation, ammonium sulfate precipitation, and chromatography on Bio-Rex 70 and phenyl-Sepharose. Renaturation after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels shows that topoisomerase activity is associated with a polypeptide with a Mr = about 111,000. The enzyme is similar to other eukaryotic type I DNA topoisomerases (nicking-closing enzymes) by the following criteria: it is capable of increasing or decreasing the topological linking number of covalently closed DNA substrate; it is capable of restoring an equilibrium distribution of linking numbers to DNA substrate with a single unique linking number; and it does not require magnesium ion or ATP for activity.     

Mutational analysis of the guanylyltransferase component of Mammalian mRNA capping enzyme

RNA guanylyltransferase is an essential enzyme that catalyzes the second of three steps in the synthesis of the 5'-cap structure of eukaryotic mRNA. Here we conducted a mutational analysis of the guanylyltransferase domain of the mouse capping enzyme Mce1. We introduced 50 different mutations at 22 individual amino acids and assessed their effects on Mce1 function in vivo in yeast. We identified 16 amino acids as being essential for Mce1 activity (Arg299, Arg315, Asp343, Glu345, Tyr362, Asp363, Arg380, Asp438, Gly439, Lys458, Lys460, Asp468, Arg530, Asp532, Lys533, and Asn537) and clarified structure-activity relationships by testing the effects of conservative substitutions. The new mutational data for Mce1, together with prior mutational studies of Saccharomyces cerevisiae guanylyltransferase and the crystal structures of Chlorella virus and Candida albicans guanylyltransferases, provide a coherent picture of the functional groups that comprise and stabilize the active site. Our results extend and consolidate the hypothesis of a shared structural basis for catalysis by RNA capping enzymes, DNA ligases, and RNA ligases, which comprise a superfamily of covalent nucleotidyl transferases defined by a constellation of conserved motifs. Analysis of the effects of motif VI mutations on Mce1 guanylyltransferase activity in vitro highlights essential roles for Arg530, Asp532, Lys533, and Asn537 in GTP binding and nucleotidyl transfer.     

Purification of wheat germ RNA ligase. II. Mechanism of action of wheat germ RNA ligase

The mechanism of action of purified wheat germ RNA ligase has been examined. ATP was absolutely required for the ligation of substrates containing 5'-OH or 5'-P and 2',3'-cyclic P or 2'-P termini. Ligation of 1 mol of 5'-P-2',3'-cyclic P-terminated poly(A) was accompanied by the hydrolysis of 1 mol of ATP to 1 mol each of AMP and PPi. Purified RNA ligase catalyzed an ATP-PPi exchange reaction, specific for ATP and dATP, and formed a covalent enzyme-adenylate complex that was detected by autoradiography following incubation with [alpha-32P]ATP and separation of the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein doublet with a molecular weight of approximately 110 kDa, the major product detected by silver staining, was labeled in these reactions. Isolated E-AMP complex was dissociated by the addition of ligatable poly(A), containing 5'-P-2',3'-cyclic P termini, to yield AMP and by the addition of PPi to yield ATP. The unique feature of the reactions leading to an exchange reaction between ATP and PPi and to the formation of an E-AMP complex was their marked stimulation (up to 400-fold) by the addition of RNA. This property distinguishes the wheat germ RNA ligase from other known RNA and DNA ligases which catalyze ATP-PPi exchange reactions and form E-AMP complexes in the absence of substrate. Thus, RNA appears to function in two capacities in the wheat germ system: as a cofactor, to stimulate the reaction of the enzyme with ATP, and as an authentic substrate for ligation.     

Eucaryotic initiation factor 4B of wheat germ binds to the translation initiation region of a messenger ribonucleic acid

Purified preparations of eucaryotic initiation factor 4B (eIF4B) from wheat germ bind the monocistronic, uncapped, mRNA satellite tobacco necrosis virus RNA (STNV RNA) in nitrocellulose-mediated binding assays. This reaction is mRNA specific and yields dissociation constants (Kd) in the 10(-7)-10(-8) M range, depending upon the particular enzyme preparation tested. Purified wheat germ eIF4A, in the presence or absence of ATP, does not bind STNV RNA efficiently, but added eIF4A and ATP do enhance the efficiency of the eIF4B-dependent binding of STNV RNA. Wheat germ eIF4B binds the oligonucleotide containing the 5'-terminal 52 nucleotides of STNV RNA (designated 1-52) with the same affinity as intact STNV RNA. This binding affinity is less with the 1-44 oligonucleotide of STNV RNA and does not occur with the 1-33 oligonucleotide of STNV RNA that contains the 5'-terminal untranslated region and the initiator AUG codon at positions 30-32 of this mRNA. Wheat germ eIF4B therefore binds the translation initiation region of STNV RNA, and this binding requires up to 20 nucleotides on the 3' side of the initiator AUG codon of this mRNA. Wheat germ eIF4B also efficiently binds an oligonucleotide containing nucleotides from positions 13-52 in from the 5' terminus of STNV RNA, thereby establishing that the postulated 5'-terminal stem and loop secondary structure of STNV RNA [Leung, D. W., Browning, K. S., Heckman, J. E., RajBhandary, U. L., & Clark, J. M., Jr. (1979) Biochemistry 18, 1361-1366] is not functional or essential for this specific binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translation of collagen messenger RNA in a cell-free system derived from wheat germ.

A cell-free system for synthesizing protein from wheat germ was used to translate the messenger RNA extracted from 16-day embryonic chick calvaria. A part of the product had properties similar to collagenous peptides and served as a substrate for prolyl hydroxylase, an enzyme specific for collagen. The level of potassium was critical for the synthesis of high molecular weight products with properties similar to pro-alpha-chains. The potassium concentration for optimal protein synthesis, as judged by maximum incorporation of [3H]proline into acid precipitable material, was considerably lower than the concentration required for the synthesis of high molecular weight collagenous peptides.     

Purification and characterization of rat liver GTP cyclohydrolase I. Cooperative binding of GTP to the enzyme

GTP cyclohydrolase I, an enzyme that catalyzes the first step in the biosynthetic pathway of tetrahydrobiopterin, has been purified about 38,000-fold to apparent homogeneity from rat liver extract with a yield of 5%. The molecular weight of the enzyme was estimated to be 300,000 by gel filtration on Ultrogel AcA 34. The purified enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position corresponding to a molecular weight of 30,000. N-terminal amino acid sequence analysis gave a single amino acid at every step of the Edman degradation up to residue 10. These results suggest that the enzyme is probably a homopolymer. The enzyme showed positive cooperativity with a Hill coefficient of 2.4 at a substrate (GTP) concentration of 10-50 microM. The Vmax value of the enzyme was 45 nmol/min.mg protein. The GTP concentration producing half-maximal velocity was 30 microM at a KCl concentration of 0.1 M. This value increased as the KCl concentration rose, without any change in Vmax or Hill number. Biosynthesis of tetrahydrobiopterin may be controlled by the intracellular level of GTP.     

Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme)

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.     

An essential surface motif (WAQKW) of yeast RNA triphosphatase mediates formation of the mRNA capping enzyme complex with RNA guanylyltransferase

Saccharomyces cerevisiae RNA triphosphatase (Cet1p) and RNA guanylyltransferase (Ceg1p) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Cet1p binding to Ceg1p stimulates the guanylyltransferase activity of Ceg1p. Here we localize the guanylyltransferase-binding and guanylyltransferase-stimulation functions of Cet1p to a 21-amino acid segment from residues 239 to 259. The guanylyltransferase-binding domain is located on the protein surface, as gauged by protease sensitivity, and is conserved in the Candida albicans RNA triphosphatase CaCet1p. Alanine-cluster mutations of a WAQKW motif within this segment abolish guanylyltransferase-binding in vitro and Cet1p function in vivo, but do not affect the triphosphatase activity of Cet1p. Proteolytic footprinting experiments provide physical evidence that Cet1p interacts with the C-terminal domain of Ceg1p. Trypsin-sensitive sites of Ceg1p that are shielded from proteolysis when Ceg1p is bound to Cet1p are located between nucleotidyl transferase motifs V and VI.     

Kinetic and thermodynamic characterization of the RNA guanylyltransferase reaction

An RNA guanylyltransferase activity is involved in the synthesis of the cap structure found at the 5' end of eukaryotic mRNAs. The RNA guanylyltransferase activity is a two-step ping-pong reaction in which the enzyme first reacts with GTP to produce the enzyme-GMP covalent intermediate with the concomitant release of pyrophosphate. In the second step of the reaction, the GMP moiety is then transferred to a diphosphorylated RNA. Both reactions were previously shown to be reversible. In this study, we report a biochemical and thermodynamic characterization of both steps of the reaction of the RNA guanylyltransferase from Paramecium bursaria Chlorella virus 1, the prototype of a family of viruses infecting green algae. Using a combination of real-time fluorescence spectroscopy, radioactive kinetic assays, and inhibition assays, the complete kinetic parameters of the RNA guanylyltransferase were determined. We produced a thermodynamic scheme for the progress of the reaction as a function of the energies involved in each step. We were able to demonstrate that the second step comprises the limiting steps for both the direct and reverse overall reactions. In both cases, the binding to the RNA substrates is the step requiring the highest energy and generating unstable intermediates that will promote the catalytic activites of the enzyme. This study reports the first thorough kinetic and thermodynamic characterization of the reaction catalyzed by an RNA capping enzyme.     

Translation of albumin messenger RNA in a cell-free protein-synthesizing system derived from wheat germ.

Purified rat liver albumin mRNA directed the synthesis of albumin in a mRNA-dependent cell-free protein-synthesizing system derived from wheat germ extracts. The [3H]leucine-labeled in vitro translation product reacted with antibodies specific for albumin and co-migrated with authentic 14C-labeled serum albumin during gel electrophoresis in the presence or absence of sodium dodecyl sufate. Higher concentrations of potassium and magnesium ions were required for the translation of albumin mRNA than for total liver mRNAs. These requirements were consistent for the purified albumin as well as when it was a component in the liver mRNA mixture. At the higher potassium or magnesium concentrations, only intact albumin molecules were synthesized, whereas lower concentrations of these ions caused the production of antibody-reactive fragments. These fragments were apparently the result of premature termination of peptide synthesis and not due to endogenous proteolytic activity.