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PubMed1989

[Substrate specificity of T4 RNA-ligase. The effect of the nucleotide composition of substrates and the size of phosphate donor on the effectiveness of intermolecular ligation].

Nucleoside 2' (3'),5'-diphosphates, dinucleotides pApA, pApC, pApU, pGpC, pCpC, pUpU (phosphate donors), and trinucleoside diphosphates, such as NpCpC, NpCpU, NpUpC, NpUpU and GpApN (N = U, C, A or G; phosphate acceptors) were used to study the substrate specificity of T4 RNA ligase. Relative efficiency of the mono- and dinucleotide donors depends on the 5'-terminal nucleoside moiety of the dinucleotide: upon ligation with the minimal phosphate acceptor GpUpC, dinucleotides pApA, pApC, and pApU are more effective than nucleotide diphosphate pAp; pGpC is more effective than pGp; efficiencies of pCpC and pCp are almost identical, and efficiency of pUpU is slightly lower than that of pUp. In relative efficiency, dinucleotide donors, varying only in 5'-terminal unit, do not correspond to mononucleotides: pApC greater than pCpC greater than pGpC and pCp greater than pUp approximately pAp much greater than pGp. The effects observed for homooligomeric substrates cannot be extra-polated on heterooligomers.

Chromatography, DEAE-Cellulose↗
PubMed2001

DNA substrates containing defined oxidative base lesions and their application to study substrate specificities of base excision repair enzymes.

Reactive oxygen species generate structurally diverse base lesions in DNA. These lesions are primarily removed by base excision repair (BER) enzymes in prokaryotic and eukaryotic cells. Biochemical properties of BER enzymes such as substrate specificity, enzymatic parameters, and action mechanisms can be best studied by employing defined oligonucleotide and DNA substrates. Currently available methods are listed to prepare defined DNA substrates containing oxidative base damage and analogs. BER enzymes for oxidative base damage are classified into two subgroups that recognize pyrimidine lesions (Endo III homologs) and purine lesions (Fpg homologs), though E. coli Fpg exhibits weak repair activity for certain pyrimidine damage. Recently, several interesting findings have been reported in relation to the substrate specificity of BER enzymes. Saccharomyces cerevisiae Endo III homologs (NTG1 and NTG2) have been shown to recognize formamidopyrimidine (Fapy) lesions that are derived from purine. Endo III and Endo VIII have a very weak activity to dihydrothymine in comparison with thymine glycol. Excision of 7,8-dihydro-8-oxoguanine by Fpg and human OGG1 is paired-base-dependent, whereas that of Fapy is essentially paired-base-independent. The repair efficiency of BER enzymes is affected by surrounding sequence contexts. In general, the sequence context effect appears to be more pronounced for Fpg homologs than Endo III homologs.

Bacterial Proteins↗
PubMed1992

The presence of 17K Mr protein, a major specific substrate for kinase C, found in the triton-insoluble fraction of synaptosome prepared from rat brain.

Cytoskeletal preparation obtained from synaptosome fractions of rat cerebrum contained the activity of kinase C, which phosphorylated 17K Mr protein endogenous to the preparation. The kinase C activity associated with the synaptosome cytoskeletons is greater in the cerebellum and hippocampus than in the cerebrum. The enhancement rates of phosphorylation of the 17K Mr protein were 293%, 544%, and 526% in the Triton X-100-insoluble fractions of synaptosomes prepared from cerebral cortex, hippocampus, and cerebellum, respectively. The 17K Mr protein was distinct from myelin basic protein (MBP) for the following reasons: 1) The electrophoretic mobility of the protein was slightly smaller than that of major MBP of rat in the polyacrylamide gel of 10-20% linear gradient, and the protein was not contained in the purified rat myelin. 2) The isoelectric point of the protein was in neutral range, whereas that of MBP was in alkaline one. 3) The 17K Mr protein did not cross-react with anti-MBP antibody. The protein was shown to be a major substrate contained in the cytoskeletal preparation of synaptosome obtained from cerebrum except for contaminating MBP. Only serine residue of the 17K Mr protein was phosphorylated by the kinase C endogenous to the preparation. The results suggest strongly that the synaptic role of protein kinase C through phosphorylation of the 17K Mr protein.

Animals↗
PubMed1990

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls.

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.

Carbohydrate Sequence↗
PubMed1995

Comparison of the specificities of p70 S6 kinase and MAPKAP kinase-1 identifies a relatively specific substrate for p70 S6 kinase: the N-terminal kinase domain of MAPKAP kinase-1 is essential for peptide phosphorylation.

xxR/KxRxxSxx sequences were phosphorylated with high efficiency by both p70 S6 kinase (p70S6K) and MAPKAP kinase-1. The best substrate for MAPKAP kinase-1 (KKKNRTLSVA) was phosphorylated with a Km of 0.17 microM, and the best substrate for p70S6K (KKRNRTLSVA) with a Km of 1.5 microM. The requirement of both enzymes for Arg/Lys at position n-5 could be partially replaced by inserting basic residues at other positions, especially by an Arg at n-2 or n-4. MAPKAP kinase-1 (but not p70S6K) tolerated lack of any residue at n-5 if Arg was present at n-2 and n-3. p70S6K (but not p90S6K) tolerated Thr at position n and absence of any residue at n + 2. The peptide KKRNRTLTV, which combined these features, was relatively selective for p70S6K having a 50-fold higher Vmax/Km than MAPKAP kinase-1. Inactivation of the N-terminal kinase domain of MAPKAP kinase-1, which is 60% identical to p70S6K, abolished activity towards all peptides tested, but the enzyme retained 30-40% of its activity if the C-terminal kinase domain was inactivated.

Amino Acid Sequence↗
PubMed2005

A high-throughput screening for phosphatases using specific substrates.

A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.

Alkaline Phosphatase↗
PubMed1998

2,6-Dimethylheptanoyl-CoA is a specific substrate for long-chain acyl-CoA dehydrogenase (LCAD): evidence for a major role of LCAD in branched-chain fatty acid oxidation.

Oxidation of straight-chain fatty acids in mitochondria involves the complicated interaction between a large variety of different enzymes. So far four different mitochondrial straight-chain acyl-CoA dehydrogenases have been identified. The physiological function of three of the four acyl-CoA dehydrogenases has been resolved in recent years especially from studies on patients suffering from certain inborn errors of mitochondrial fatty acid beta-oxidation. The physiological role of long-chain acyl-CoA dehydrogenase (LCAD) has remained obscure, however. The results described in this paper provide strong evidence suggesting that LCAD plays a central role in branched-chain fatty acid metabolism since it turns out to be the major acyl-CoA dehydrogenase reacting with 2,6-dimethylheptanoyl-CoA, a metabolite of pristanic acid, which itself is the alpha-oxidation product of phytanic acid.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed1984

7S RNA, containing 5S ribosomal RNA and the termination stem, is a specific substrate for the two RNA processing enzymes RNase III and RNase E.

The 7S RNA, a precursor of 5S rRNA that contains 5S rRNA and the termination stem and loop, is a substrate for RNase E and is also a substrate for RNase III. The cleavage by RNase III is in the stem, 11 nucleotides downstream from the 3' end of the mature 5S rRNA and 8 nucleotides downstream from the RNase E cleavage site. Near the cleaved nucleotides there are three base pairs that appear in the same relative positions in most known RNase III cleavage sites. The large product of the RNase III cleavage reaction, which is a 5S rRNA that contains 11 extra nucleotides at the 3' end, is a substrate for RNase E. This suggests that the information for the 3'-end cleavage by RNase E resides mainly in the 5S rRNA itself. Using rnc rne strains, carrying the plasmid that leads to the accumulation of 7S RNA, we showed that the 7S RNA does not result from an RNase III cleavage but is apparently a proper transcription termination product.

Base Sequence↗
PubMed1997

14,15-Dehydroleukotriene A4: a specific substrate for leukotriene C4 synthase.

We studied the metabolism of 14,15-dehydro-leukotriene A4 (14, 15-dehydro-LTA4) by human platelet leukotriene C4 (LTC4) synthase and polymorphonuclear leucocyte (PMNL) leukotriene A4 (LTA4) hydrolase. Metabolites were separated and identified using reversed-phase HPLC coupled to diode-array UV detection. Human platelets metabolize 14,15-dehydro-LTA4 to 14,15-dehydro-LTC4 with apparent kinetics identical with authentic LTA4. Metabolism to 14, 15-dehydro-LTC4 is inhibited by MK-886, a reported LTC4 synthase inhibitor in human platelets, with a potency comparable with that shown by LTA4. In contrast, neither human red-blood-cell lysates nor human PMNL enzymically convert 14,15-dehydro-LTA4 into 14, 15-dehydro-leukotriene B4. Minor amounts of 14,15-dehydro-LTC4, observed in some PMNL preparations, result from variable eosinophil contamination, as confirmed using highly purified neutrophil and eosinophil-enriched preparations. In addition, 14,15-dehydro-LTA4 irreversibly inhibits PMNL LTA4 hydrolase with an IC50 of 0.73 microM. The geometry of the methyl terminus of LTA4 does not influence the metabolism by human platelet LTC4 synthase. The double bond at C-14,15 is essential for the catalytic activity of LTA4 hydrolase but not for binding to this enzyme.

Binding Sites↗
PubMed1999

Product of side-chain cleavage of cholesterol, isocaproaldehyde, is an endogenous specific substrate of mouse vas deferens protein, an aldose reductase-like protein in adrenocortical cells.

Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5-6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD(50). In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis.

Adrenal Cortex↗
PubMed2000

Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme.

The endoplasmic reticulum enzyme UDP-glucose glycoprotein:glucosyltransferase (UGGT) has the unique property of recognizing incompletely folded glycoproteins and, if they carry an N -linked Man(9)GlcNAc(2)oligosaccharide, of catalyzing the addition of a glucose residue from UDP-glucose. Using peptide sequence information, we have isolated the complete cDNA of rat liver UGGT and expressed it in insect cells. The cDNA specifies an open reading frame which codes for a protein of 1527 residues including an 18 amino acid signal peptide. The protein has a C-terminal tetrapeptide (HEEL) characteristic of endoplasmic reticulum luminal proteins. The purified recombinant enzyme shows the same preference for unfolded polypeptides with N -linked Man(9)GlcNAc(2)glycans as the enzyme purified from rat liver. A genetically engineered Saccharomyces cerevisiae strain capable of producing glyco-proteins with Man(9)GlcNAc(2)core oligosaccharides was constructed and secreted acid phosphatase (G0-AcP) was purified. G0-AcP was used as an acceptor glycoprotein for UGGT and found to be a better substrate than the previously used soybean agglutinin and thyroglobulin. Recombinant rat UGGT has a K (m) of 44 microM for UDP-glucose. A proteolytic fragment of UGGT was found to retain enzymatic activity thus localizing the catalytic site of the enzyme to the C-terminal 37 kDa of the protein. Using site-directed mutagenesis and photoaffinity labeling, we have identified residues D1334, D1336, Q1429, and N1433 to be necessary for the catalytic activity of the enzyme.

Acid Phosphatase↗
PubMed1991

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity.

A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.

Amino Acid Sequence↗
PubMed2005

Two glutathione peroxidases in the fungal pathogen Cryptococcus neoformans are expressed in the presence of specific substrates.

Glutathione peroxidases catalyse the reduction of peroxides by reduced glutathione. To determine if these enzymes are important for resistance to oxidative stress and evasion of the innate immune system by the fungal pathogen Cryptococcus neoformans, two glutathione peroxidase homologues, which share 38 % identity, were identified and investigated. In this study, these peroxidases, Gpx1 and Gpx2, their localization, their contribution to total glutathione peroxidase activity, and their importance to the oxidative and nitrosative stress resistance of C. neoformans are described. It is shown that the two glutathione peroxidase genes are differentially expressed in response to stress. While both GPX1 and GPX2 are induced during t-butylhydroperoxide or cumene hydroperoxide stress and repressed during nitric oxide stress, only GPX2 is induced in response to hydrogen peroxide stress. Deletion mutants of each and both of the glutathione peroxidases were generated, and it was found that they are sensitive to various peroxide stresses while showing wild-type resistance to other oxidant stresses, such as superoxide and nitric oxide. While the glutathione peroxidase mutants are slightly sensitive to oxidant killing by macrophages, they exhibit wild-type virulence in a mouse model of cryptococcosis.

Animals↗
PubMed1994

The role of 80K/MARCKS, a specific substrate of protein kinase C, in cell growth and tumour progression.

Since its discovery more than a decade ago [Wu et al., 1982; Rozengurt et al., 1983], the 80-87 kDa myristoylated alpha lanine-rich C-kinase substrate (80K/MARCKS) protein has attracted a great deal of attention from researchers interested in cell growth and tumour progression. However, despite its ubiquitous distribution, a definitive functional role for 80K/MARCKS has not been found. The purpose of this review is to describe the properties, distribution and regulation of 80K/MARCKS and to discuss some of the most recent findings, both from our laboratory and from others, that have suggested a functional role for this protein in modulating cell growth and tumour progression. Furthermore, I will present data from our laboratory that implicates 80K/MARCKS as a novel tumour suppressor in cells of melanocyte origin.

Actins↗