Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with $\text{bis-(}\text{p-}\text{nitrophenyl)}$ phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards $\text{5′ -}\text{dTMP}\text{-p-}\text{nitrophenyl}$ ester, which is probably associated with intracellular membrane structure in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

A substrate for the fluorogenic assay of exo-beta-N-acetylmuramidase: synthesis and purification of 4-methylumbelliferyl N-acetylmuramide.

A fluorogenic substrate for exo-β-N-acetylmuramidase from Bacillus subtilis B was synthesized. 4-Methyl-2-oxo-1,2-benzopyran-7-yl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside was prepared from 4-methyl-2-oxo-1,2-benzopyran-7-yl 2-acetamido-2-deoxy-β-d-glucopyranoside, condensed with dl-2-chloropropionic acid, the benzylidene residue removed by acetolysis and the 4-methyl-2-oxo-1,2-benzopyran-7-yl 2-amino-3-O-(d-1-carboxyethyl)-2-deoxy-β-d-glucopyranoside purified by chromatography on silica gel and Sephadex G-10 and by high-voltage paper electrophoresis. The identity of the product was confirmed by pmr studies, acid hydrolysis followed by chromatography of the products, and enzymic digestion.     

Enzymatic synthesis of p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside as a substrate for human alpha-amylases.

Enzymatic modification at the nonreducing end D-glucosyl residue of p-nitrophenyl alpha-maltopentaoside was developed by using the transglycosylation of beta-D-galactosidase from Bacillus circulans. The enzyme regioselectively synthesized p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside (a yield of 12.0% based on the amount of p-nitrophenyl alpha-maltopentaoside added) on a preparative scale from lactose as a donor and p-nitrophenyl alpha-maltopentaoside as an acceptor. It revealed that the nonreducing end galactosyl group of p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside did not prohibit the action of human salivary and pancreatic alpha-amylases. This derivative was shown to be very suitable as a novel substrate for analytical use of human alpha-amylase assay in serum through a conjugated reaction involving glucoamylase and alpha-D-glucosidase.     

Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.     

Continuous photometric determination of endo-1,4-beta-D-glucanase (cellulase) activity using 4-methylumbelliferyl-beta-D-cellobioside as a substrate

A very simple and sensitive procedure for the determination of the activity of highly purified endo-1,4-beta-glucanase from the microscopic fungus Trichoderma reesei using 4-methylumbelliferyl-beta-D-cellobioside has been developed. The HPLC study has shown that this substrate is cleaved by endo-1,4-beta-glucanase to form predominantly free 4-methylumbelliferone, Km and kcat being 1.25 mM and 7.9 s-1, respectively (30 degrees C, pH 5.0). The possibility of continuous photometric determination of the enzyme using the difference absorptivity coefficient of 1600 M-1 cm-1 at 350 nm has been demonstrated.     

An ultrasensitive, continuous assay for xylanase using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside

We describe a fluorescence-based assay for the analysis of xylanase activity using a novel fluorogenic substrate, 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside (DiFMUX(2)). Generation of fluorescent 6,8-difluoro-4-methylumbelliferone (DiFMU) from the substrate corresponded directly to xylanase activity. High-performance liquid chromatography analysis of the digestion products showed that xylanase hydrolyzed DiFMUX(2) directly to DiFMU and xylobiose. The assay provides the speed, sensitivity, and convenience required for measuring xylanase activity or for screening xylanase inhibitors in a high-throughput format and is suitable for the kinetic assay of xylanases from a variety of sources.     

Acetyl phosphate as a substrate for the calcium ATPase of sarcoplasmic reticulum

Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt.     

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U     

n-Propanol as a substrate for assaying the ligninperoxidase activity of Phanerochaete chrysoporium.

The steady state kinetics of ligninperoxidase catalysed reaction using n-propanol as the organic substrate and monitoring the formation of propanaldehyde at lambda = 300 nm spectrophotometerically as functions of different reaction parameters has been studied. It has been concluded that n-propanol can be used as a substrate for analysing the activity of ligninperoxidase. The turnover number of ligninperoxidase of Phanerochaete chrysosporium using n-propanol as substrate has been found to be higher approximately by a factor of 10(3) as compared to that using veratryl alcohol as the substrate. The method works in assaying the activity of ligninperoxidase produced by Aspergillus fumigatus indicating that it can be used for assaying the ligninperoxidase activities produced by other microorganisms also and is not limited to assaying the ligninase activity produced by Phanerochaete chrysosporium alone. Under identical experimental conditions, horseradish peroxidase does not show peroxidase activity using n-propanol as substrate indicating that the method does not interfere with the activities of other peroxidases.     

A comparison of the activity of phosphatidylinositol phosphodiesterase against substrate in dispersions and as monolayers at the air-water interface.

The activity of phosphatidylinositol phosphodiesterase, purified from rat brain, against substrate in three forms, (a) multibilayer liposomes, (b) single bilayer vesicles of phosphatidylinositol and (c) phosphatidylinositol oriented as monolayers at the air-water interface, was examined. The reaction rate was similar against the two substrate dispersions prepared with the same phospholipid concentration, although there was a large difference in substrate surface area available to the enzyme, and this similarity could not be accounted for by any differences in the microviscosity of the hydrocarbon region of the phospholipid bilayers. The reaction showed apparent zero-order reaction kinetics until about 10% of the substrate had been degraded, whereupon the rate decreased. The reaction against monolayers of phosphatidylinositol was linear throughout the entire digestion of the film, provided that more than 0.25 mg of enzyme was present in the subphase. The pH optimum was 6.6. Bivalent ions )Ca2+, Mg2+, Co2+, Ni2+ and Mn2+) facilitated enzyme penetration into substrate monolayers, but the enzyme was only activated by Ca2+ (optimal concentration, 1mM) and to a lesser extent by Mg2+. The reaction rate was independent of initial surface pressures of less than about 22mN-m(-1) but at higher pressures the rate decreased. This decrease could be prevented by the addition of 10mol of octadecylamine/90mol of phosphatidylinositol to the substrate monolayer; the amine did not increase the rate of reaction in films of less than 22mN-m(-1).     

Synthesis of 2-n-(hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide, a chromogenic substrate for assaying sphingomyelinase activity.

2-N-(Hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide a compound resembling sphingomyelin is synthesized. It is cleaved by sphingomyelinase to the chromogenic N-acylaminonitrophenyl moiety. Phospholipase C preparations do not hydrolyze this compound. The starting material is 2-amino-4-nitrophenol which when acylated with palmitoyl chloride yields the hexadecananilide. Reaction with beta-bromoethylphosphoryldichloride gives the phosphate which is quaternized with trimethylamine to give the title compound.     

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin.

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.     

CaMKII uses GTP as a phosphate donor for both substrate and autophosphorylation

The vast majority of serine/threonine protein kinases have a strong preference for ATP over GTP as a phosphate donor. CK2 (Casein kinase 2) is an exception to this rule and in this study we investigate whether calcium/calmodulin-dependent protein kinase II (CaMKII) has the same extended nucleotide range. Using the Drosophila enzyme, we have shown that CaMKII uses Mg²⁺GTP with a higher K_m and V_max compared to Mg²⁺ATP. Substitution of Mn²⁺ for Mg²⁺ resulted in a much lower K_m for GTP, while nearly abolishing the ability of CaMKII to use ATP. These similar results were obtained with rat αCaMKII, showing the ability to use GTP to be a general property of CaMKII. The V_max difference between Mg²⁺ATP and Mg²⁺GTP was found to be due to the fact that ADP is a potent inhibitor of phosphorylation, while GDP has modest effects. There were no differences found between sites autophosphorylated by ATP and GTP, either by partial proteolysis or mass spectrometry. Phosphorylation of fly head extract revealed that similar proteins are substrates for CaMKII whether using Mg²⁺ATP or Mg²⁺GTP. This new information confirms that CaMKII can use both ATP and GTP, and opens new avenues for the study of regulation of this kinase.