Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor

Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.     

Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates

A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS. The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely beta-structure by circular dichroism. The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11. The enzyme was more labile in Tris-HCI buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more. The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint).     

Expression and characterization of frog peptidylglycine alpha-hydroxylating monooxygenase

We report here a recombinant Chinese hamster ovary cell system,which is able to stably express frog peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3), the first enzyme responsible for the formation of peptide C-terminal amide. This system excreted PHM mostly into the medium and almost no PHM activity was detected in the cell lysate. Three differentiation inducers were examined to determine whether or not they would enhance the PHM expression. Addition of 4mM sodium butyrate into the medium increased the expression of PHM activity about 4-fold at 48 h after addition. Increases of about 2-fold were observed in the cases of sodium propionate or N,N(')-hexamethylene-bis-acetamide. Through a three-step purification procedure, we obtained 5mg purified PHM, which showed a single band at 40 kDa on SDS-PAGE, from 2-L of conventional monolayer culture medium. The reactions with three synthetic substrates, D-Tyr-Val-Gly, N-trinitrophenyl-D-Tyr-Val-Gly (TNPYVG), and hippuric acid (HA), were characterized. Of these, TNPYVG was the most active substrate. The pH optima for TNPYVG and HA were pH 5-6, while that for D-Tyr-Val-Gly was pH 7.5. There is a possibility that the substrate N-terminal structure may affect the interaction between the substrate and the enzyme catalytic site.     

Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate

Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 mM Hepes or phosphate buffer, pH 7.5, 100 microM PK-1, 100 microM [gamma-32P]ATP, 3 mM MgCl2, 12 mM KCl, and 20-200 ng of catalytic subunit assayed at 30 degrees C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.     

5'-Nucleotidase from rat heart

5'-Nucleotidase has been extracted from rat heart and purified to apparent homogeneity. The enzyme is a glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the apparent molecular weight of the subunit is 74 000 at several different gel concentrations. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows that the enzyme is a dimer. The enzyme hydrolyzes all nucleoside 5'-monophosphates tested. A comparison of Vmax/Km for 14 different substrates shows that AMP is the best substrate. The enzyme shows lowest Km values for AMPS, AMP, isoAMP, GMP, and IMP. It shows no activity with nucleoside 2'- and 3'-monophosphates, sugar phosphates, and p-nitrophenyl phosphate, even when tested at high enzyme concentrations. The optimum activity of the enzyme occurs at pH 7.5 with AMP as substrate. Above this pH, buffer ions affect the activity in a complex manner, a second optimum being observed under some conditions. Magnesium ions activate the enzyme above pH 7.5 in the presence of some buffer ions but not of others. Magnesium ions show only a slight activation when the reaction is run in diethanolamine buffer, pH 9.5, at 30 degrees C; the activation in this buffer is considerably greater when the reaction is run at 37 degrees C. The enzyme is strongly inhibited by free ADP, maximum inhibition occurring below pH 6. The ADP inhibition is diminished as the pH is raised above 6, becoming negligible above pH9. The enzyme is inhibited by EDTA. The inhibition is partially reversed when the EDTA is removed from the enzyme by gel filtration. This as well as other evidence indicates that the enzyme contains a tightly bound metal ion.     

Beef kidney 3-hydroxyanthranilic acid oxygenase. Purification, characterization, and analysis of the assay.

Beef kidney 3-hydroxyanthranilic acid oxygenase has been purified to homogeneity. It is a single subunit protein of Mr = 34,000 +/- 2,000 with a frictional coefficient (f/f0) of about 1.1. The enzyme readily aggregates to form, apparently inactive, higher molecular weight oligomers. The very rapid loss of enzyme activity during the assay was analyzed extensively. It was found to be due to inactivation of the enzyme by the substrate, 3-hydroxyanthranilate, and unrelated to enzyme turnover or oxidation of bound iron. The loss of activity was shown to be a first order decay process, and methods are given for obtaining accurate initial reaction rates under all conditions. Evidence was presented that the enzyme assumes a catalytically inactive conformation at pH 3.4, which only relatively slowly rearranges to an active form at pH 6.5; the rearrangement can be blocked by the presence of substrate. We have found that Fe2+, which is required for enzymatic activity, can equilibrate freely, albeit slowly, with the enzyme during the course of the enzyme reaction even in the presence of saturating 3-hydroxanthranilate. Under assay conditons, the Fe2+ has an apparent dissociation constant of 0.04 mM. The kinetic properties of the enzyme were found to be dramatically different in beta,beta-dimethylglutarate buffer and collidine buffer; both the rate of loss of activity during the assay and the substrate Km and Vmax were affected.     

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification, properties, and kinetics of the tyrosine-sensitive isoenzyme from Escherichia coli.

The tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate-phosphorylating), EC 4.2.1.15) was purified to homogeneity from extracts of Escherichia coli K12. A spectrophotometric assay of the enzyme activity, based on the absorption difference of substrates and products at 232 nm, was developed. The enzyme has a molecular weight of 66,000 as judged by gel filtration on Sephadex G-200, and a subunit molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This suggests either a rapid monomer-dimer equilibrium, or a very asymmetric shape for the native enzyme. The enzyme shows a narrow pH optimum around pH 7.0. The enzyme is stable for several months when stored at -20 degrees in phosphate buffer containing phosphoenol-pyruvate. Intersecting lines in double reciprocal plots of initial velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism with-catalyzed reaction. Product inhibition studies specify an ordered sequential BiBi mechanism with a dead-end E-P complex. The feedback inhibitor tyrosine at concentrations above 10 muM exhibits noncompetitive inhibition with respect to erythrose-4-P, and competitive inhibition with respect to the other substrate, P-enolpyruvate. In addition, tyrosine at concentrations of at least 10 muM causes an alteration of one or more than one kinetic parameter of the enzyme.     

A microchip-based enzyme assay for protein kinase A.

A microchip-based enzyme assay for protein kinase A is described. The microchips were prepared by standard photolithographic techniques. The assay reagents were placed in wells on the microchips, and electroosmosis was used to transport aliquots of these reagents into the network of etched channels, where the enzymatic reaction takes place. Protein kinase A catalyzes the transfer of a phosphate group from ATP to the serine residue of the heptapeptide LeuArgArgAlaSerLeuGly (Kemptide). The outcome of the enzymatic reaction was assessed by performing an on-chip electrophoretic separation of the fluorescently labeled peptide substrate and product. All liquid-handling steps were performed by controlling the electroosmotically driven flow from reagent and buffer wells using electrical current. On-chip dilutions of the peptide substrate, ATP and H-89, a known protein kinase A inhibitor, were performed and the kinetic constants (K(m), K(i)) of these compounds were determined. This prototype assay demonstrates the usefulness of the microchips for performing enzymatic assays for which fluorogenic substrates cannot easily be designed.     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.     

A biochemical study of non-specific esterases from plant cells, employing the histochemical substrate, naphthol AS-D acetate

Synopsis The reagents routinely employed in the histochemical detection of nonspecific esterases, namely naphthol AS-D acetate and Fast Red Violet LB salt, have been used to study these enzymes biochemically with spectrophotometric procedures. A range of parameters that affect the coupling of the hydrolyzed substrate and diazonium salt were examined and their relevance to future histochemical procedures is noted.TheK _m of broad-bean root-tip esterases was estimated to be approximately 0.07 mM, but other kinetic data suggest that the true value is lower. From an analysis of the kinetics of the hydrolytic reaction it appears that they are of a mixed nature over the time course and substrate concentrations used.The effect of pH on root tip esterase activity has been examined and the recorded optimum of 5.5 is similar to that reported by other workers for plant cells. Non-enzymic hydrolysis of the substrate at alkaline pH levels prevented measurement of enzyme activity beyond pH 7.2.Magnesium ions at a final concentration of 5 mM are important in retaining esterases in their natured state during enzyme extraction, although the addition of increasing amounts of the ion to the assay system caused a corresponding increase in esterase inhibition.     

Engineering sensitive glutathione transferase for the detection of xenobiotics

Cytosolic glutathione transferases (GSTs) are a major reserve of high-capacity ligand binding proteins which recognise a large variety of hydrophobic compounds. In the present study, the binding of non-substrate xenobiotic compounds (herbicides and insecticides) to maize GST I was investigated by employing kinetic inhibition studies, site-directed mutagenesis and molecular modelling studies. The results showed that the xenobiotics bind at the substrate binding site. Based on in silico docking analysis, two residues were selected for assessing their contribution to xenobiotic binding. The mutant Gln53Ala of GST I Exhibits 9.2-fold higher inhibition potency for the insecticide malathion, compared to the wild-type enzyme. A potentiometric assay was developed for the determination of malathion using the Gln53Ala mutant enzyme. The assay explores the ability of the xenobiotic to promote inhibition of the GST-catalysing 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH) conjugation reaction. The sensing scheme is based on the pH change occurring in a low buffer system by the GST reaction, which is measured potentiometrically using a pH electrode. Calibration curve was obtained for malathion, with useful concentration range 0-20muM. The method's reproducibility was in the order of +/-3-5% and malathion recoveries were 96.7+/-2.8%. Immobilized Gln53Ala mutant GST was used to assemble a biosensor for malathion. The enzyme was immobilized by crosslinking with glutaraldehyde and trapped behind a semipermeable membrane in front of the pH electrode. The results demonstrated that the immobilized enzyme behaved similar to free enzyme.     

Inhibition of beta-lactamase of Bacillus licheniformis 749/C by compound PS-5, a new beta-lactam antibiotic.

By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.     

Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase

Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42 degrees C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k(cat)) for methylation was 88 and 32 h(-1) on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 ((K(m)(pep))) were 0.9 and 1.0 microM and for S-adenosyl-L-methionine (K(m)(AdoMet)) were 1.8 and 0.6 microM, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di- to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.     

Further characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary

The ability of purified bovine neurointermediate pituitary peptidyl glycine alpha-amidating monooxygenase to catalyze the conversion of peptide substrates (D-Tyr-X-Gly) into amidated product peptides (D-Tyr-X-NH2) was evaluated. The pH optimum of the reaction was pH 8.5 when X was Val, Trp, or Pro, but 5.5 to 6.0 when X was Glu. Similar maximum velocity (Vmax) values were obtained for the Val, Trp, and Pro substrates while the Glu substrate had a substantially higher Vmax. The Michaelis-Menten constant (Km) of the enzyme for the peptide substrate increased in the order Trp less than Val less than Pro much less than Glu. Increasing levels of ascorbate brought about parallel increases in Km and Vmax, suggesting the presence of an irreversible step separating the interaction of the enzyme with the two substrates. The effect of copper on enzyme activity was dependent on the peptide substrate and the reaction pH. With the Val substrate, exogenous copper was required for optimal activity; no other metal ion tested could substitute for copper. With the Glu substrate, exogenous copper was not required for optimal activity; however, diethyldithiocarbamate, a copper chelator, inhibited activity and only copper could reverse this inhibitory effect. The ability of various cofactors to stimulate alpha-amidating monooxygenase activity was also dependent on assay conditions. With the Val or Glu substrate in the presence of exogenous copper, a variety of cofactors in addition to ascorbate were capable of supporting activity. With the Glu substrate in the absence of exogenous copper, the requirement of the enzyme for ascorbate was more strict. In keeping with the proposed reaction mechanism, nearly 1 mol ascorbate was consumed for each mole of D-Tyr-Glu-NH2 produced.     

UDPglucosyltransferase and its kinetic fluorimetric assay

A rapid, kinetic assay for UDPglucosyltransferase has been developed using 1-naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1-naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ. Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP-glucuronic acid, UDP-N-acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1-naphthol and UDP-glucose have also been determined.     

A direct spectrophotometric assay for peptide deformylase.

A direct UV-VIS spectrophotometric assay has been developed for peptide deformylase. This assay employs a novel class of peptide mimetics as deformylase substrates which, upon enzymatic removal of the N-terminal formyl group, rapidly release free thiols. The released thiols are quantitated using Ellman's reagent. A variety of peptide analogues that contain beta-thiaphenylalanine or beta-thiamethionine as the N-terminal residue were synthesized and found to be excellent substrates of the peptide deformylase from Escherichia coli (k(cat)/K(M) = 6.9 x 10(5) M(-1) s(-1) for the most reactive substrate). The deformylase reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion. The versatility of the assay has been demonstrated by its application to kinetic characterization of the deformylase, pH profile studies, and enzyme inhibition assays. The assay can also be performed in an end-point fashion. The results demonstrate that this assay is a simple, highly sensitive, and rapid method to study kinetic properties of deformylases without the use of any coupling enzymes.     

The effect of pH on the kinetics of beef-liver fructose bisphosphatase.

1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of MN2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate.     

The synthesis, purification, and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases: design of a substrate based on subsite preferences

A convenient chromophoric assay for porcine pepsin has been developed using a new synthetic substrate. The sequence of this substrate was chosen based on the known subsite preferences for this enzyme. The peptide contains a phenylalanyl-p-nitrophenylalanine sequence at the reactive site. Cleavage of this bond yields a change in absorbance at 310 nm of between 1700 and 2000 per mole. This allows kinetic data to be obtained readily and accurately. The products of cleavage have been identified by isolation of a peptide fragment by high-performance liquid chromatography. Values of kcat, Km, and kcat/Km of 94 +/- 6 s-1, 0.13 +/- .04 mM, and 815 +/- 210 s-1/mM-1 were obtained at pH 3.0 and 37 degrees C. The peptide is soluble over the pH range from 2 to 7, thus facilitating determination of the pH dependence of the kinetic parameters. The substrate is also valuable in studying the inhibition of pepsin.     

High-performance liquid chromatographic assay for acid and alkaline phosphatase in serum

High-performance liquid chromatography was used to assay serum acid and alkaline phosphatase. Samples were incubated with adenosine-5′-monophosphoric acid (AMP) in a buffer of required pH, 5′-nucleotidase was inhibited with Ni²⁺ ions, and the phosphatase activity was determined by measuring the concentration of the reaction product, adenosine. The analysis time, after the incubation is terminated, is short (7 min), and the assay is quantitative and reproducible. Complete separation of the reaction product from the substrate and the naturally occurring serum constituents and the high sensitivity of the ultra-violet detection system eliminate some of the problems commonly encountered in spectrophotometric assays.