Authentic Substrate Research Articles

Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays.

The Rieske Fe/S protein of the cytochrome b6/f complex in chloroplasts: missing link in the evolution of protein transport pathways in chloroplasts?

The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b(6)/f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the DeltapH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH(2)-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of DeltapH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways.

A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta.

We have developed a method of general application for identifying putative substrates of protein kinases in cell extracts. Using this procedure, we identified the physiological substrates of several mitogen-activated protein kinase kinases and an authentic substrate of stress-activated protein kinase (SAPK) 2a/p38. A 120 kDa protein was detected in skeletal muscle extracts that was phosphorylated rapidly by SAPK4/p38delta, but poorly by SAPK2/p38, SAPK3/p38gamma, SAPK1/JNK or extracellular signal-regulated kinase 2 (ERK2). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38delta phosphorylated eEF2K at Ser359 in vitro, causing its inactivation. eEF2K became phosphorylated at Ser359 and its substrate eEF2 became dephosphorylated (activated) when KB cells were exposed to anisomycin, an agonist that activates all SAPKs, including SAPK4/p38delta. The anisomycin-induced phosphorylation of Ser359 was unaffected by SB 203580, U0126 or rapamycin, and was prevented by overexpression of a catalytically inactive SAPK4/p38delta mutant, suggesting that SAPK4/p38delta may mediate the inhibition of eEF2K by this stress. The phosphorylation of eEF2K at Ser359 was also induced by insulin-like growth factor-1. However, this was blocked by rapamycin, indicating that Ser359 is targeted by at least two signalling pathways.

Dialysis and ultrafiltration of molasses for fermentation enhancement

The pore structure of a commercially available dialysis membrane was modified by the deposition of crystals of calcium octanoate in the membrane matrix. The modified structure resulted in a significant reduction in sucrose loss when dialysis was used to reduce the potassium concentration of a solution of sucrose and potassium chloride. The results of fermentation studies of artificial molasses substrates using a proprietary strain of Saccharomyces cerevisiae showed that the optimum potassium concentration for micro-organism activity was ∼10.5 g l−1. Subsequent fermentation studies on an authentic Australian molasses substrate with an initial potassium concentration (11.0 g l−1) close to the optimum value showed the predicted decrease in micro-organism activity following potassium removal using the modified membrane. However, molasses from most Australian sources has a potassium concentration substantially higher than the optimum value when diluted for use as a substrate and in that case fermentation performance would be expected to be enhanced by potassium removal. Also, it was shown that the rate of fermentation of molasses could be increased by macromolecule removal using an ultrafiltration membrane with a nominal molecular weight cut-off of ∼300 000 Da.

Engineering subtilisin E for enhanced stability and activity in polar organic solvents.

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).

17] Purification of yeast mitochondrial chaperonin 60 and co-chaperonin 10

Publisher Summary The chapter presents a study on the purification of yeast mitochondrial chaperonin 60 and co-chaperonin 10. The chapter describes a large-scale purification procedure of S. cerevisiae hsp60 expressed in Pichia pastoris. Yeast cpnl0 was isolated by taking advantage of the fact that bacterial GroEL forms a functional complex with co-chaperonins from mitochondria or chloroplasts. Chaperonin system is not involved in the folding of all mitochondrial proteins. Assaying the interaction of purified hsp60 and cpnl 0 with authentic substrate proteins should give insight into the specificity of the mitochondrial chaperonin system. In addition, a comparison of the bacterial GroEL/ES system with the eukaryotic hsp60/cpnl0 system may help to elucidate the chaperonin reaction cycle. The chapter describes the procedure for purification of recombinant heat shock protein 60, purification of recombinant authentic and hexahistidine-tagged chaperonin 10, and also mentions the characterization of purified yeast chaperonins.

Homologous regions of the alpha subunit of eukaryotic translational initiation factor 2 (eIF2alpha) and the vaccinia virus K3L gene product interact with the same domain within the dsRNA-activated protein kinase (PKR).

The vaccinia virus K3L gene product, pK3, binds to the dsRNA-activated protein kinase, PKR, reducing its ability to interact with and phosphorylate eIF2alpha. On the basis of this characteristic and the homology of pK3 to the N-terminus of eIF2alpha, several laboratories have utilized pK3 to investigate the molecular determinants that specify substrate recognition by PKR. The data presented here demonstrate that the natural substrate, eIF2alpha, also binds to PKR in vitro and interacts with the same or an overlapping domain within PKR. A truncated form of eIF2alpha, representing the N-terminal 123 amino acids and containing the regions of homology to pK3, retains the ability to bind PKR. pK3, eIF2alpha, and the truncated form of eIF2alpha all bind to the C-terminus of PKR containing the catalytic domain, but not to the regulatory N-terminus. Variants of pK3 and eIF2alpha, des-(75-78)-K3L (pK3deltaGYID), and des-(80-83)-eIF2alpha (eIF2alphadeltaGYID), from which the conserved amino acids GYID have been deleted, exhibit a decreased ability to interact with PKR. Similarly, the in vitro binding of pK3, eIF2alpha, and the truncated form of eIF2alpha to PKR can be competed with purified pK3 but not with pK3deltaGYID. In addition, the deletion of GYID from eIF2alpha significantly reduces its ability to be phosphorylated by PKR, demonstrating that PKR recognizes its substrate, at least in part through interaction with sequences remote from the phosphorylation site. In summary, we have shown that the region within PKR that interacts with the pseudosubstrate, pK3, is the same region that interacts with the authentic substrate, eIF2alpha. In addition, we have shown that the N-terminal 123 amino acids of eIF2alpha contains structural elements necessary for recognition by PKR. The results pinpoint the GYID motif, shared between pK3 and eIF2alpha and distant from the phosphorylation site, as being important for the interaction of eIF2alpha with PKR, as well as its phosphorylation.

Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha.

The mammalian double-stranded RNA-activated protein kinase PKR is a component of the cellular antiviral defense mechanism and phosphorylates Ser-51 on the alpha subunit of the translation factor eIF2 to inhibit protein synthesis. To identify the molecular determinants that specify substrate recognition by PKR, we performed a mutational analysis on the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. High-level expression of PKR is lethal in the yeast Saccharomyces cerevisiae because PKR phosphorylates eIF2alpha and inhibits protein synthesis. We show that coexpression of vaccinia virus K3L can suppress the growth-inhibitory effects of PKR in yeast, and using this system, we identified both loss-of-function and hyperactivating mutations in K3L. Truncation of, or point mutations within, the C-terminal portion of the K3L protein, homologous to residues 79 to 83 in eIF2alpha, abolished PKR inhibitory activity, whereas the hyperactivating mutation, K3L-H47R, increased the homology between the K3L protein and eIF2alpha adjacent to the phosphorylation site at Ser-51. Biochemical and yeast two-hybrid analyses revealed that the suppressor phenotype of the K3L mutations correlated with the affinity of the K3L protein for PKR and was inversely related to the level of eIF2alpha phosphorylation in the cell. These results support the idea that residues conserved between the pseudosubstrate K3L protein and the authentic substrate eIF2alpha play an important role in substrate recognition, and they suggest that PKR utilizes sequences both near and over 30 residues from the site of phosphorylation for substrate recognition. Finally, by reconstituting part of the mammalian antiviral defense mechanism in yeast, we have established a genetically useful system to study viral regulators of PKR.

Assay methods for methyl farnesoate esterases in crustaceans

Methyl farnesoate (MF) is a sesquiterpenoid that is synthesized by crustaceans and is structurally similar to the insect juvenile hormones. MF is metabolized to farnesoic acid by carboxylesterases present in crustacean tissues. In order to investigate the biological significance of MF metabolism, we have developed two rapid methods for measuring MF esterase activity. The first method is a radiochemical partition assay that utilizes an authentic substrate. The [3H]MF partition assay was used to evaluate the spectrophotometric esterase substrates methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-pentylthioacetothioate (PENTAT) for use with crude and partially purified samples of MF esterases from lobster hepatopancreas (midgut gland). The spectrophotometric method is less specific for MF esterases than the partition assay but is less time consuming and nonradioactive, and it provides kinetic information. HEPTAT and PENTAT were suitable for rapid screening of chromatographic fractions for MF esterase activity. Arch. Insect Biochem. Physiol. 36:115–128, 1997. © 1997 Wiley-Liss, Inc.

Lipids and lipolytic enzymes in the trunkwood ofRobinia pseudoacacia L. during heartwood formation

The radial profile of lipase and phospholipase activities was determined in the trunkwood of Robinia pseudoacacia L. The trees were felled in November at the time of heartwood formation and alterations in the enzymatic activities were investigated across the sapwood and heartwood. Methods employed include gaschromatographic, colorimetric and enzymatic assays. On a dry weight basis, the hydrolysis of the artificial substrate p- nitrophenylpalmitate shows a maximum activity in growth ring 4; however, the assay has proved not to be specific for lipase. In contrast, lipase analyses (triacylglycerol acylhydrolase; E.C. 3.1.1.3) with an authentic substrate show activity peaks in growth rings 1 and 4. With protein as a reference the highest activity is found in growth ring 5. A similar tendency is observed for phospholipase A l (E.C. 3.1.1.32) and phospholipase A2 (E.C. 3.1.1.4). Phospholipase C (E.C. 3.1.4.3) activity decreases towards the sapwood-heartwood boundary; negligible traces of activity are detected in the heartwood, whereas, based on the protein content, growth ring 4 yields maximal activity. Phospholipase D (3.1.4.4) exhibits the same radial pattern with regard to protein content as a reference. On a dry weight basis there is a significant increase within the sapwood area, while in the heartwood the activity drastically decreases. The enzyme activities are discussed in relation to degradative processes within the plasma membranes and the hydrolysis of reserve lipids during heartwood formation.

Lipids and lipolytic enzymes in the trunkwood ofRobinia pseudoacacia L. during heartwood formation

The radial profile of lipase and phospholipase activities was determined in the trunkwood ofRobinia pseudoacacia L. The trees were felled in November at the time of heartwood formation and alterations in the enzymatic activities were investigated across the sapwood and heartwood. Methods employed include gaschromatographic, colorimetric and enzymatic assays. On a dry weight basis, the hydrolysis of the artificial substrate pnitrophenylpalmitate shows a maximum activity in growth ring 4; however, the assay has proved not to be specific for lipase. In contrast, lipase analyses (triacylglycerol acylhydrolase; E.C. 3.1.1.3) with an authentic substrate show activity peaks in growth rings 1 and 4. With protein as a reference the highest activity is found in growth ring 5. A similar tendency is observed for phospholipase A1 (E.C. 3.1.1.32) and phospholipase A2 (E.C. 3.1.1.4). Phospholipase C (E.C. 3.1.4.3) activity decreases towards the sapwood-heartwood boundary; negligible traces of activity are detected in the heartwood, whereas, based on the protein content, growth ring 4 yields maximal activity. Phospholipase D (3.1.4.4) exhibits the same radial pattern with regard to protein content as a reference. On a dry weight basis there is a significant increase within the sapwood area, while in the heartwood the activity drastically decreases. The enzyme activities are discussed in relation to degradative processes within the plasma membranes and the hydrolysis of reserve lipids during heartwood formation.

Polylysine and CVIM Sequences of K-RasB Dictate Specificity of Prenylation and Confer Resistance to Benzodiazepine Peptidomimetic in Vitro

BZA-5B, a benzodiazepine peptidomimetic, inhibits CAAX farnesyltransferase (FTase) and blocks attachment of farnesyl groups to oncogenic and wild-type H-Ras in animal cells. This compound slows the growth of cells transformed with oncogenic H-Ras at concentrations that do not affect the growth of nontransformed cells. This finding suggested that nontransformed cells may produce a form of Ras whose prenylation is resistant to BZA-5B. In the current studies, we found that FTase had a 50-fold higher affinity for K-RasB than for H-Ras in vitro. Farnesylation of K-RasB was inhibited by BZA-2B, the active form of BZA-5B, but only at concentrations that were 8-fold higher than those that inhibited farnesylation of H-Ras. K-RasB, but not H-Ras, was also a substrate for CAAX geranylgeranyltransferase-1 (GGTase-1), and its affinity for the enzyme was equal to that of Rap1B, an authentic leucine-terminated substrate for GGTase-1. Inhibition of the geranylgeranylation of K-RasB occurred only at high concentrations of BZA-2B. All of these properties of K-RasB were traced to the combined effects of its COOH-terminal CVIM sequence and the adjacent polylysine sequence, neither of which is present in H-Ras. These studies provide a potential explanation for the resistance of nontransformed cells to growth inhibition by BZA-5B. Inasmuch as the majority of Ras-related human cancers contain oncogenic versions of K-RasB rather than H-Ras, the current data suggest that in vitro studies of FTase inhibitors with potential anti-cancer activity should use authentic K-RasB as a substrate.

Human urinary kallikrein can generate angiotensin II from homologous renin substrates.

We previously proposed the "kinin-tensin system," a unique vasoregulatory system that can produce both angiotensin II and kinins. To verify whether tissue kallikrein is a part of this system in humans, we examined the ability of human urinary kallikrein (HUK) to generate angiotensin (ANG) II directly from homologous renin substrates such as purified human angiotensinogen (AOGEN) and authentic human tridecapeptide renin substrate (13 RS). HUK released ANG II not only from ANG I but also directly from both AOGEN and 13RS at an optimum pH of 7.0. The amount of generated ANG II from 7.5 nmol of each of the three substrates at pH 7.0 was as follows: ANG I, 292.7 +/- 67.2; 13 RS, 1951.7 +/- 239.6; AOGEN, 2.2 +/- 0.3 (pmol/3h, n = 3 mean +/- SE). HUK cleaved Phe-His and His-Leu bonds in 13 RS, and Tyr-Ile and Phe-His bonds in ANG I. These results suggest that HUK is a part of the "kinintensin system", i.e., HUK can not only release kinins, but can also generate ANG II mainly through ANG I conversion and from AOGEN, the latter being a minor source of ANG II. Furthermore, HUK may play a role in regulating vascular tone under certain conditions in vivo.

Specificity of dopachrome tautomerase and inhibition by carboxylated indoles. Considerations on the enzyme active site

Dopachrome tautomerase (EC 5.3.2.3) catalyses the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) within the melanin-formation pathway. We have analysed a series of substrate analogues and related compounds as possible substrates and inhibitors of tautomerization. The enzyme appears to be highly specific since D-dopachrome, alpha-methyldopachrome, dopaminochrome, adrenochrome methyl ether and deoxyadrenochrome are not substrates. Conversely, dopachrome tautomerase catalyses the tautomerization of dopachrome methyl ester, suggesting that a carboxy group, either free or as a methyl ester, is essential for enzyme recognition. No inhibition of dopachrome tautomerization was observed in the presence of either semiquinonic compounds, such as tropolone and L-mimosine, or pyrrole-2-carboxylic acid and unsubstituted indole. However, a number of indole derivatives, including DHICA, the product of dopachrome tautomerization, and the analogues 5-hydroxyindole-2-carboxylic and indole-2-carboxylic acid were able to inhibit the enzyme. Furthermore, indoles with a side chain at position 3 of the ring and containing a carboxylic group at the gamma-position of this chain, such as L-tryptophan or indole-3-propionic acid, are stronger inhibitors of the enzyme. Indole-3-carboxylic acid, indole-3-acetic acid and indole-3-butyric acid are very weak inhibitors, showing that the carboxylic group needs to be located at an optimal distance from the indole ring to mimic the carboxylic group at position 2 on the authentic substrate.

Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli

The 3C region of human rhinovirus 14 was expressed in Escherichia coli. The microbially synthesized protease was functional, since the expressed precursor underwent autoproteolytic processing to generate mature molecules of the expected molecular weight and antigenicity. Mutation of the putative active-site Cys-146 residue to an alanine resulted in the synthesis of unprocessed precursor molecules. Large quantities of the 20-kilodalton protease were purified by a simple purification protocol, and the resulting molecule was shown to be biologically active in vitro against synthetic peptides corresponding to the 2C-3A cleavage site. This site was cleaved with high efficiency and fidelity and was used to generate kinetic data on the 3C protease. The protease exhibited sensitivity to Zn2+, was capable of cleaving five of seven rhinovirus cleavage site peptides tested with variable efficiency, and could distinguish authentic substrate peptides from control peptides containing the dipeptide cleavage sequence pair Gln-Gly.

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.

Mechanism of salicylate hydroxylase-catalyzed decarboxylation

Salicylate hydroxylase (salicylate, NADH: oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.1) in Pseudomonas putida catalyzed hydroxylation of the substrate analogue, salicylaldehyde, to form catechol and formate with stoichiometric consumption of NADH and O 2. Consequently, a study of primary product derived from the carboxyl group of the authentic substrate, salicylate, was undertaken. The experimental results revealed that CO 2 not H 2CO 3, was produced first.

Comparison of various β-glucosidase assays used to diagnose gaucher's disease

Gaucher's diselse is an inborn error of metabolism in which glucocerebroside accumulates in cells of the reticuloendothelial system due to a marked deficiency in the activity of lysosomal glucocerebrosidase. Five different published fluorometric assay procedures that utilize 4-methylumbelliferyl-β-D-gluco-pyranoside as the substrate to determine β-glucosidase activity in fibroblasts, leukocytes and liver from patients and heterozygote carriers of Gaucher's disease were compared with the results obtained using a glucocerebrosidase assay that employed authentic radiolabeled glucocerebroside as the substrate. When cultured skin fibroblasts were used as a source of enzyme, fluorometric β-glucosidase assays performed either at pH 4–4.05 in the absence of sodium taurocholate or at pH 5.4–5.5 in the presence of the bile salt, all proved effective in confirming the diagnosis of Gaucher's disease and in identifying heterozygous carriers of the disease. The fluorometric β-glucosidase assay procedures that were most effective in evaluating relative glucocerebrosidase activity in leukocytes from patients and carriers of Gaucher's disease were those that included sodium taurocholate in the incubation medium. When human liver biopsy served as the source of enzyme, only the glucocerebrosidase assay employing authentic glucocerebroside substrate proved effective in confirming the diagnosis of Gaucher's disease: fluorometric assays performed at pH 4 or pH 5.4–5.5 in the presence or absence of the bile salt sodium taurocholate do not always identify patients with Gaucher's disease.