Use of fluorescamine-labeled casein as a substrate for assay of proteinases.

Conditions have been investigated for the use of fluorescamine-labeled casein as a substrate for fluorometric assay of proteinases. Fluorescamine-labeled casein can be prepared simply by mixing solutions of casein and fluorescamine at pH 8.0 and used without removal of the excess reagent or its hydrolysis product. The fluorescence of the labeled casein and its enzymatic digest is moderately stable in the range of pH 7.0 to 10.0. Activities can be determined by measuring the fluorescence of the hydrolysis products soluble in 0.1 M trichloro acetic acid solution at pH 4.0 after adjusting the pH of the acid-soluble fraction to 7.7. This method is suited for assay of proteinases active at neutral to slightly alkaline pH values, and is capable of quantitating about 0.05 microgram of trypsin or 0.5 microgram of alpha-chymotrypsin or papain. The assay can be done in the presence of large amounts of contaminating amino acid, protein and/or exopeptidases which may interfere with the ordinary assay of proteinases.     

Comparative electrophoretic analysis of human and porcine plasminogen activators in SDS-polyacrylamide gels containing plasminogen and casein

Electrophoretic analysis of plasminogen activators from pig heart, human uterus, human plasma and human melanoma cells was performed in SDS-polyacrylamide gradient slab gels containing plasminogen and casein. Direct visualization of activator activity bands in polyacrylamide gels was achieved after removal of SDS, incubation in buffer, and staining with Coomassie brilliant blue. Tissue activator extracted from pig hearts displayed a molecular weight of 72000 and migrated similarly to activator secreted by human melanoma cells and to one activator component present in extracts of human uterus. Immunoadsorption experiments with melanoma cell activator antiserum indicated that these 72-kDa activators are all related immunologically. Human uterus also contained a second activator component with a molecular weight 55000, which migrated similarly to a higher molecular weight component of urokinase and cross-reacted with urokinase antiserum. We conclude that the 72-kDa uterine activator component represents a tissue activator and the 55-kDa component represents a urokinase-like activator. A euglobulin solution from venous occlusion plasma displayed multiple bands of plasmin activity in the Mr range 85000-96000. Two activator components were also present, one of Mr 72000 and another of Mr 62000. The 72-kDa euglobulin activator was adsorbed by MCA antiserum, and we conclude that this component represents vascular activator. The 62000 activator also had weak plasminogen-independent caseinolytic activity and was not affected by either melanoma cell activator or urokinase antisera. Conclusions concerning its identity cannot be made at this time.     

A new assay of X-prolyl dipeptidyl-aminopeptidase activity in human serum with glycylproline p-phenylazoanilide as substrate

Summary A new assay procedure for X-prolyl dipeptidyl-aminopeptidase activity in human serum was developed with glycylproline p-phenylazoanilide tosylate as substrate. p-Phenylazoaniline liberated by the enzyme reaction was measured photometrically at 493 nm after stopping the reaction with acid. This assay was simple and sensitive, and less than 50 l of human serum was required for the assay. Km value was 2.5 mm and the optimum pH was 8.7. After disc gel electrophoresis of human serum, the enzyme activity could be distinctly observed as a reddish band on the gel when the gel was incubated with this substrate.     

Phosphate groups as substrate determinants for casein kinase I action

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates.     

Role of acidic residues as substrate determinants for casein kinase I

Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase.     

Identity and substrate specificity of human erythrocyte membrane-bound and cytosolic casein kinases

The relationship and substrate specificity of the human erythrocyte membrane kinase and casein kinase A were investigated. Based on Staphylococcus aureus V8 protease digestion patterns, the 2 kinases appeared to be structurally homologous. These enzymes also exhibited the same substrate specificity and phosphorylated the same synthetic peptides and domains of ankyrin. Both kinases did not utilize GTP effectively as a substrate and were not inhibited by low concentrations of heparin, suggesting that they were type I casein kinases. An analysis of synthetic peptide phosphorylation failed to reveal a specific pattern of recognition of the amino acid sequence surrounding the phosphorylation site.     

Epoxide hydratase assay in human platelets using hepoxilin A3 as a lipid substrate

1-14C-labelled hepoxilin A3 (8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid) was generated from 1-14C-labelled arachidonic acid during incubation with a rat lung preparation lacking epoxide hydratase activity. The HPLC purified hepoxilin A3 gave only two isomeric 8,11,12-triols (termed trioxilins A3) upon incubation with a rat lung preparation containing epoxide hydratase activity. Based on this simple reaction an assay was developed using only 2000 cpm/tube of substrate and aliquots of a homogenate of platelet membranes from man. Products were assayed by thin-layer radiochromatography. Males were noted to have higher epoxide hydratase activity for this substrate than females.     

The use of indole as a spectrophotometric assay substrate for toluene dioxygenase

Abstract Cell extracts of toluene-grown Pseudomonas putida produced a soluble yellow dye during aerobic incubations with indole and NADH. Accumulation of indoxyl in reaction mixtures corresponded with a linear increase in absorbance at 400 nm. The rate of increase in absorbance was shown to be a specific measure of toluene dioxygenase activity. The primary product of toluene oxidation, cis -toluene dihydrodiol, inhibited dioxygenase activity in cell extracts containing no detectable activity of cis -toluene dihydrodiol dehydrogenase.     

Real-time fluorogenic kinase assay using protein as substrate

A real-time fluorogenic kinase assay using myelin basic protein (MBP) as a substrate is reported. MBP is part of a noncovalent complex with a negatively charged, dye-labeled lipopeptide, (N-heptadecanoyl)-K(dye2)-linker-EEIYGEF-amide. The complex is approximately 20 times less fluorescent than the free lipopeptide. The MBP-lipopeptide complex serves as a protein substrate for several Ser/Thr kinases. We infer that the observed fluorescence increase on the addition of kinase and ATP is due to the phosphorylation of MBP, which decreases the affinity of MBP with the negatively charged, dye-labeled lipopeptide. Several protein kinases (protein kinase C βII, mitogen-activated protein kinase [MAPK] Erk1, and MAPK Erk2) were tested with the assay. The assay exhibited a fivefold fluorescence increase over background, provided kinetic values comparable to literature values (apparent K_m^ATP), and produced inhibitor constants comparable to literature values for a typical inhibitor, namely staurosporine.     

Hyaluronidase assay using fluorogenic hyaluronate as a substrate

The reducing terminal of hyaluronate was labeled with a fluorogenic reagent, 2-aminopyridine. The pyridylaminohyaluronate was incubated with testicular hyaluronidase for 1 h. After incubation, 4 vol of ethanol was added to the incubation mixture, followed by centrifugation. The fluorescence of the supernatant containing the degradation products of hyaluronidase digestion was then determined by fluorospectrophotometry (excitation wavelength, 320 nm; emission wavelength, 400 nm). It was found that the increase of the pyridylamino products was linearly correlated with enzyme concentration (up to 0.1 national formulary unit), incubation time (up to 60 min), and substrate concentration (up to 2.5 microM). The fluorogenic substrate was also applicable for the determination of crude hyaluronidase. This simple, rapid, and sensitive hyaluronidase assay was made possible by the use of pyridylaminohyaluronate as a substrate.     

Evaluation of ultrasound velocity measurements for estimating protease activities using casein as substrate

Ultrasonic resonator technology (URT) was compared with the well established UV–Vis/ninhydrin assay to estimate protease activities in defined buffer systems. Hydrolysis of casein was measured using subtilisin, trypsin, halophilic protease from Haloferax mediterranei and Bacillus lentus alkaline protease. Sensitivity, reproducibility, working range as well as the limit of detection and the limit of quantification were comparable for both methods. Salt concentrations (0.5 M NaCl) interfered with the URT method. The quantification of protease activity by URT was possible when the product concentration measured by the UV–Vis/ninhydrin assay was correlated to the corresponding ultrasonic velocity signals.     

Chemiluminescent assay of alkaline phosphatase using dihydroxyacetone phosphate as substrate detected with lucigenin

A sensitive and simple chemiluminescent assay (CL) for alkaline phosphatase (ALP) using dihydroxyacetone phosphate or its ketal (DHAP or DHAP‐ketal) was developed. New substrates were transformed to dihydroxyacetone (DHA) after they were hydrolysed by ALP, which reacts with lucigenin and produces strong chemiluminescence. Under the optimum assay condition, the detection limits were 3.8 × 10^?19 and 1.5 × 10^?18 moles of ALP, respectively. The coefficients of variation (CV) at each points on the standard curve were 0.8–5.4% and 1.8–7.1% (n = 6), respectively. The mechanism of lucigenin CL with DHA was investigated by ESR spectrometry using the spin‐trapping method. The mechanism was speculated as follows: the O₂^? generated by the reaction of DHA and O₂ in alkaline solution reacts with lucigenin, and then emit light. The proposed CL assay was applied to the enzyme immunoassay of 17β‐oestradiol, using ALP as a label enzyme. The measurable range of 17β‐oestradiol was 15–4000 pg/mL, and the proposed method was four times more sensitive than the colorimetric assay for ALP by using 4‐nitrophenyl phosphate as substrate. Copyright © 2002 John Wiley & Sons, Ltd.     

Interaction of casein with human polymorphonuclear cells

Attachment of 125I-casein to PMN cells was investigated. Iodination did not decrease the chemotactic effect of casein. 125I-casein binding was increasing toward a maximum reached at about 45 min at 24, and 37 degrees C. At 4 degrees C the binding was proportional to time for 45 min. No saturation was achieved even at 15 mg/ml casein. About 40% of casein remained attached to PMN in a casein-free medium after 60 min, at 37 degrees C. Pretreatment of the cells with trypsin or butanol, or the presence of indomethacin, azide, and PMSF did not affect the binding of casein. The hydrophobic amino acid, leucin counteracted the attachment of casein. Our data show that at chemotactic doses casein is bound specifically to cell membranes by hydrophobic forces. The induction of chemotaxis may be due to micellar casein-membrane lipid complexes.     

Comparison of casein of cynomolgus monkey (Macaca fascicularis) with human casein

Casein of cynomolgus monkey was compared with those from human and bovine milk. Cynomolgus monkey casein showed similar electrophoretical patterns to those of human casein on Disc- and SDS-electrophoresis. It consisted of beta- and kappa-casein-like components. The component corresponding to bovine alpha s1-casein was not detected. The beta-casein-like fraction of cynomolgus monkey showed 9 bands on Disc-PAGE. These were suggested to be the same protein binding different levels of phosphorus by dephosphorylation experiment using an acid phosphatase. The kappa-casein-like component of cynomolgus monkey was highly glycosylated (about 50% carbohydrate) similarly as human kappa-casein and the constituent carbohydrates were same as those detected in human kappa-casein (galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid). Amino acid composition of cynomolgus monkey kappa-casein bore a resemblance to those of both human and bovine kappa-caseins. Amino acid composition of cynomolgus monkey beta-casein was also similar to those of human and bovine beta-caseins.