Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

Substrate activity screening (SAS) is a fragment-based method for the rapid development of novel substrates and their conversion into non-peptidic inhibitors of Cys and Ser proteases. The method consists of three steps: (i) a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay; (ii) the identified N-acyl aminocoumarin substrates are optimized by rapid analog synthesis and evaluation; and (iii) the optimized substrates are converted into inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. This protocol describes a general procedure for the solid-phase synthesis of a library of N-acyl aminocoumarin substrates and the screening procedure to identify weak binding substrates.     

Molecular modeling of the binding of 5-substituted 2'-deoxyuridine substrates to thymidine kinase of herpes simplex virus type-1

In an earlier study, De Winter and Herdewijn (J. Med. Chem. 1996, 37, 4727-4737) studied the binding of various 5-substituted 2'-deoxyuridine substrates to thymidine kinase of herpes simplex virus type-1. They used a computational procedure that achieves good correlation with experimentally determined IC50 values. We applied an alternative procedure to the same deoxyuridine substrates, using only three readily calculated quantities-the binding energy, the molecular surface area, and a flexibility factor. Our simplified method achieves the same degree of correlation with the IC50 values as did the earlier procedure. We then applied this procedure to examine the binding of various 5-substituted pyrimidine 1,5-anhydrohexitol substrates to thymidine kinase.     

Rapid Determination of Norepinephrine, Dopamine, Serotonin, Their Precursor Amino Acids, and Related Metabolites in Discrete Brain Areas of Mice Within Ten Minutes by HPLC with Electrochemical Detection

A rapid and simple chromatographic procedure using HPLC with electrochemical detection is described for simultaneous determination of the substrates from precursor amino acids to metabolites related to synthesis and metabolism of three monoamine neurotransmitters--norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin)--in discrete brain areas of the mouse. Under the present instrumental and mobile phase conditions, the procedure permits simultaneous determination of three monoamines (NE, DA, and 5-HT), two precursor amino acids (tyrosine and tryptophan), and four respective metabolites (3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid) within 10 min in one chromatographic run. By varying column temperature, this procedure also permits simultaneous determination of 10-14 monoamine-related substrates including the nine substrates described above within 15-21 min. The validity of the present procedure is demonstrated by analyzing the effect of an alpha 2-adrenergic agonist (clonidine) and an alpha 2-antagonist (yohimbine) in mouse hypothalamus.     

Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C

An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.     

Modification of the 14C most-probable-number method for use with nonpolar and volatile substrates.

A method was developed to allow the use of volatile and nonpolar substrates in 14C most-probable-number tests. Naphthalene or hexadecane was sorbed to filter paper disks and submerged in minimal medium. The procedure reduced the volatilization of the substrates while allowing them to remain available for microbial degradation.     

Modification of the 14C most-probable-number method for use with nonpolar and volatile substrates

A method was developed to allow the use of volatile and nonpolar substrates in 14C most-probable-number tests. Naphthalene or hexadecane was sorbed to filter paper disks and submerged in minimal medium. The procedure reduced the volatilization of the substrates while allowing them to remain available for microbial degradation.     

Preparation and use of metal surface-immobilized DNA hairpins for the detection of oligonucleotides

The following protocol describes the experimental steps used to prepare arrayable and label-free biological sensors that are based on the fluorescence unquenching of DNA hairpins immobilized on metal surfaces. This two-part protocol describes both the creation of gold-coated substrates and the oligonucleotide surface self-assembly process that transforms the substrates into reporters for detecting DNA. Using this procedure, one can create sensors for oligonucleotides that are highly sensitive and have demonstrated an exceptional specificity to single nucleotide polymorphisms. From start to finish, the entire procedure can be accomplished in 24-30 h.     

Prediction of cyclin-dependent kinase phosphorylation substrates

Protein phosphorylation, mediated by a family of enzymes called cyclin-dependent kinases (Cdks), plays a central role in the cell-division cycle of eukaryotes. Phosphorylation by Cdks directs the cell cycle by modifying the function of regulators of key processes such as DNA replication and mitotic progression. Here, we present a novel computational procedure to predict substrates of the cyclin-dependent kinase Cdc28 (Cdk1) in the Saccharomyces cerevisiae. Currently, most computational phosphorylation site prediction procedures focus solely on local sequence characteristics. In the present procedure, we model Cdk substrates based on both local and global characteristics of the substrates. Thus, we define the local sequence motifs that represent the Cdc28 phosphorylation sites and subsequently model clustering of these motifs within the protein sequences. This restraint reflects the observation that many known Cdk substrates contain multiple clustered phosphorylation sites. The present strategy defines a subset of the proteome that is highly enriched for Cdk substrates, as validated by comparing it to a set of bona fide, published, experimentally characterized Cdk substrates which was to our knowledge, comprehensive at the time of writing. To corroborate our model, we compared its predictions with three experimentally independent Cdk proteomic datasets and found significant overlap. Finally, we directly detected in vivo phosphorylation at Cdk motifs for selected putative substrates using mass spectrometry.     

Histochemistry of carboxyl esterases in the broad bean root tip with indoxyl substrates

Summary The possibility of the use of indoxyl substrates for enzyme localization in plant material was tested and proved. Studying the localization of the carboxyl esterase in broad bean root tip a) the time course of the indigogenic reaction was investigated, b) the concentration of the ferro/ferri redox buffer was examined, c) various substances were used to control the indigogenic visualizing reaction, d) the azocoupling procedure was applied with indoxyl substrates, e) unhalogenated and halogenated substrates were tried, and, finally, f) acetate and butyrate were employed in the indigogenic procedure. Using the last mentioned (f) trial the existence and localization was verified in the materal tested of carboxyl esterase fractions recognized there previously on the basis of azocoupling reactions with naphtholic substrates.     

A mass spectrometry-based method for the assay of ceramide synthase substrate specificity

The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.24). Ceramide contains a long-chain base (LCB) linked to a variety of fatty acids to produce a lipid class with potentially hundreds of structural variants. An optimized procedure for the assay of ceramide synthase in yeast microsomes is reported that uses mass spectrometry to detect any possible LCB and fatty acid combination synthesized from unlabeled substrates provided in the reaction. The assay requires the delivery of substrates with bovine serum albumin for maximum activity within defined limits of substrate concentration and specific methods to stop the reaction and extract the lipid that avoid the non-enzymatic synthesis of ceramide. The activity of ceramide synthase in yeast microsomes is demonstrated with the four natural LCBs found in yeast along with six saturated and two unsaturated fatty acyl-coenzyme As from 16 to 26 carbons in length. The procedure allows for the determination of substrate specificity and kinetic parameters toward natural substrates for ceramide synthase from potentially any organism.     

Use of 13C substrates for metabolic studies in exercise: methodological considerations

The purpose of this study is to outline a common mistake made when the rate of oxidation of exogenous substrates during prolonged exercise is computed using 13C naturally labeled substrates. The equation proposed and commonly used in the computation does not take into account that exercise and/or exogenous substrate ingestion modifies the composition of the mixture of endogenous substrates oxidized and, consequently, the isotopic composition of CO2 arising from oxidation of endogenous substrates. The recovery of 13C and the amount of exogenous substrate oxidized are thus overestimated. An adequate procedure for the computation of exogenous substrate oxidation taking into account changes in isotopic composition of CO2 arising from oxidation of endogenous substrates is suggested. Results from a pilot experiment (4 subjects) using this procedure indicate that over 2 h of exercise (66% of maximal O2 uptake), with ingestion of 60 g of glucose, 39 +/- 4 g of glucose were oxidized. Estimates made without taking into account changes in isotopic composition of CO2 arising from oxidation of endogenous substrates range between 70 +/- 8 and 44 +/- 3 g depending on 1) the isotopic composition of exogenous glucose and 2) the isotopic composition of expired CO2 taken as reference (rest or exercise without glucose ingestion). These observations suggest that results from previous studies of exogenous substrate oxidation during exercise using 13C labeling should be used with caution.     

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.     

Inhibition of adenosine 3':5'-monophosphate-dependent protein kinase: comparison of a protein inhibitor with polyanions and substrate analogs.

The mechanism of inhibition of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase was studied using a protein inhibitor isolated by a non-denaturing procedure from bovine heart. This protein inhibitor interacts with the catalytic subunit of protein kinase and binds to some substrates of the kinase. Protein kinase activity can also be inhibited by polyanions which, like the protein inhibitor, bind to basic substrates but do not bind to the catalytic subunit of protein kinase. Peptides such as L-lysyl-L-tyrosyl-L-threonine that resemble the phosphate accepting site of protein kinase substrates competitively inhibit phosphorylation of histone. Protein kinase activity can thus be inhibited in vitro by interaction of the protein inhibitor with substrates, and/or the catalytic subunit of the kinase, by competition of substrate analogs with "natural" substrates and by direct interaction of polyanions with basic protein substrates for the phosphotransferase reaction.     

A microtiter plate procedure for evaluating fungal functional diversity on nitrogen substrates

Ascertaining the effects of anthropogenic disturbance on belowground diversity is of paramount importance because pollution from agricultural practices and industrialization are increasing worldwide. Although we have methods for evaluating soil microbial function with respect to carbon use our ability to evaluate use of other compounds is limited. Because N cycling is of paramount importance in ecosystem stability, evaluation of the ability of saprophytic soil fungi to use a variety of N sources would provide important information on possible alterations in ecosystem stability with disturbance. Herein is described a procedure (soil Nitrolog) for evaluating fungal functional diversity on a suite of 95 different N substrates. The soil Nitrolog procedure was evaluated by testing fungal functional diversity at two sites in Big Bend National Park (Chihuahuan Desert), differing in elevation and plant community composition. The soil Nitrolog procedure distinguished between the two sites based on overall use of the 95 N substrates. In addition the procedure detected differences in individual substrate use based on site specific plant compounds in response to changes in the amount of N entering these ecosystems from anthropogenic inputs.     

Assay of L-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.     

An assay for acidic peptide substrates of protein kinases

The assay of acidic peptides as substrates for protein kinases has not been as easy to perform as testing basic peptides or polypeptides. We have developed a simple, rapid, and cost-effective procedure that allows the design and testing of potential peptide substrates without the constraints imposed by the phosphocellulose filter paper method (the need to incorporate positively charged residues into the peptide sequence). The technique combines the chelation of 32Pi by acid molybdate with PEI-cellulose chromatography. In this way the migration of 32P-labeled Pi, ATP, and protein are impeded while phosphopeptide is eluted in 1.5 ml from a 0.25-ml disposable column. In order to validate the assay we used two angiotensin II analogues as peptide substrates for the protein tyrosine kinase pp60c-src. The assay results using the new procedure were compared to those of the phosphocellulose filter paper technique. We also demonstrated the use of this method to test linear and cyclic peptides that could not be assayed with the phosphocellulose paper technique. This assay will aid those who are attempting to determine the substrate specificity of protein kinases.     

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.     

A simple assay for methionine adenosyltransferase using cation exchange paper and liquid scintillation spectrometry

A rapid, sensitive and simple procedure for the assay of l-methionine-S-adenosyltransferase based on the use of phosphocellulose ion-exchange paper is presented. The analytical procedure may be generally useful for all enzymes where there is a large cationic charge difference between the substrates and products. An application of a simpllified counting procedure for radioisotopes on solid supports is presented.     

A new family of D-2-hydroxyacid dehydrogenases that comprises D-mandelate dehydrogenases and 2-ketopantoate reductases

The gene for the D-mandelate dehydrogenase (D-ManDH) of Enterococcus faecalis IAM10071 was isolated by means of an activity staining procedure and PCR and expressed in Escherichia coli cells. The recombinant enzyme exhibited high catalytic activity toward various 2-ketoacid substrates with bulky hydrophobic side chains, particularly C3-branched substrates such as benzoylformate and 2-ketoisovalerate, and strict coenzyme specificity for NADH and NAD(+). It showed marked sequence similarity with known NADP-dependent 2-ketopantoate reductases (KPR). These results indicate that together with KPR, D-ManDH constitutes a new family of D-2-hydroxyacid dehydrogenases that act on C3-branched 2-ketoacid substrates with various specificities for coenzymes and substrates.     

Lactic acid production from molasses utilizing Lactobacillus delbrueckii and invertase together

Conclusion The prices of the process substrates such as glucose, sucrose and molasses (as $/ton) are 1500, 1600 and 24, respectively. For molasses plus invertase, the price increases to 46 $/ton. Thus compared with the other possible substrates, the lactic acid production procedure used in this study does not cause any appreciable increase in the pruduction cost due to the utilization of invertase, while enhancing the yield of product.