Reaction rate measurements of proteases and glycosidases with chromogenic methods

Abstract

Simultaneous azo-coupling and indigogenic methods were evaluated for the quantitative histochemical assay of the plasma membrane proteases gamma-glutamyl transpeptidase (EC 2.3.2.2) and dipeptidyl peptidase IV (EC 3.4.14.5) and the glycosidases maltase-glucoamylase and glucoamylase (EC 3.2.1.20) in decidual cells, jejunal enterocytes and renal proximal tubulocytes. Using kinetic (continuous) microdensitometry, a linear increase in the final reaction product was found from 3 up to 10 min, depending on the substrate concentration and the plasma membrane glycosidase or protease under investigation. Combined continuous and end point (static) microdensitometry revealed a linear relationship between the section thickness (enzyme concentration) and final reaction product up to 12 microns for the proteases and up to 16 microns for the glycosidases. Apparent Km and Vmax values were calculated with a computerized version of the direct linear plot and compared with the results obtained with the linear transformations according to Lineweaver-Burk, Eadie-Hofstee and Hanes. Apparent Km and Vmax values for the proteases were calculated separately for each animal and were 1.82 mM and 1.02 mM and 2.43 arbitrary units (a.u.) and 1.67 a.u. (gamma-glutamyl transpeptidase, decidua) and 0.42 mM and 0.38 mM and 0.29 and 0.26 a.u. (dipeptidyl peptidase IV, decidua). For the alpha-D-glucosidases, the corresponding values were 0.23 mM and 0.15 a.u. (kidney) and 0.55 mM and 0.20 a.u. (jejunum). The results show the suitability of the indigogenic methods for quantitative histochemical measurements of plasma membrane alpha-D-glucosidases, whereas the simultaneous azo-coupling procedures seemed to be less suitable for the quantification of surface membrane proteases, due to, for example, interactions of diazonium salts with amino acid or peptide substrates, reaction products and peptide activators.

Estimating the initial reaction velocity of a soluble dehydrogenase in situ

“…One reason for the difficulty is that the activities of many enzymes, particularly dehydrogenases, do not increase linearly with time during the initial stages of their incubation in tissue sections. Examples include LDH in heart (Altman, 1978;Frederiks et al, 1989;Van Noorden & Vogels, 1989), skeletal muscle (Nolte & Pette, I972b;Altman, 1978), liver (Nolte & Pette, 1972a;Stoward & Nakae, 1988;Frederiks et al, (Altman, 1978); succinate dehydrogenase in heart (Altman, 1978;Van Noorden & Vogels, 1989), skeletal muscle (Nolte & Pette, 1972b;Pette, 1981), liver (Nolte & Pette, 1972a;Stoward & Nakae, 1988;Nakae & Stoward, 1992a) and motor neuron (Chalmers & Edgerton, 1989); gluc0se-6-phosphate dehydrogenase in liver (Butcher & Van Noorden, 1985;Van Noorden et al, 1985;Van Noorden & Butcher, 1987); glyceraldehyde-3-phosphate dehydrogenase in skeletal muscle (Nolte & Pette, 1972b); 3]3-hydroxysteroid dehydrogenase in ovary (Robertson et aI., 1982;Lomax eta]., 1989); 20~-hydroxysteroid dehydrogenase in ovary (Robertson et al, 1984); NADPH-ferrihaemoprotein reductase in liver (Van Noorden & Butcher, 1986); alkaline phosphatase in liver (Van Noorden & Jonges, 1987); NADH tetrazolium reductase in skeletal muscle (Lomax et al, 1989); y-glutamyl transpeptidase and dipeptidyl peptidase IV in decidua (Ruhnke & Gossrau, 1989); and low-Kin hexokinase in submandibular gland (Lawrence eta]., 1989). Four theories have been proposed for the initial non-linearity of dehydrogenase catalysed reactions in situ.…”

Fluorescent Assays To Quantitate Enzymatic Activities Yielding as End Product an Aqueous-Insoluble Indigo-Blue Dye

“…Indoxyl-derivatized substrates are available for hydrolytic enzymes such as sialidase, − β-galactosidase, glucosidase, esterase, ,,, protease, glycosidase, sulfatase, lipase, glucuronidase, and phosphatase .…”

“…The various enzymes acting on the respective substrates produced an aqueous-insoluble indigo-blue dye for visual, histochemical, or diagnostic detection. The 4,7-di- O -methyl-α- d - N -acetyl-neuraminic acid, specific for influenza virus types A and B neuraminidases, was used in the ZstatFlu Test for the clinical diagnostic of influenza. , While the utility of such techniques is significant, quantitative measurement of enzymatic activity required time-consuming, labor-intensive, and often imprecise methods such as TLC, scanning densitometry, or organic solvent dissolution of indigo-blue for visible absorption spectroscopy. , Two quantitative fluorescent techniques are described in this paper for influenza viral neuraminidase activity toward indoxyl-derivatized substrates.…”

“…Due to indigo-blue's poor solubility, quantitative enzyme kinetics that liberates the dye is difficult to perform. Techniques such as microdensitometry were used to determine the enzyme kinetic constants ( K m , V max ); however, such techniques are labor intensive, time-consuming, and often imprecise. Two methods are described in this paper to quantitatively measure sialidase/neuraminidase activity toward three indigogenic substrates: 5-bromo-indol-3yl α- d - N -acetyl-neuraminic acid, 5-bromo-indol-3-yl 4,7-di-O-methyl-α- d - N -acetyl-neuraminic acid, and 5‘-bromo-4-chloro-3-indolyl-α- d - N -acetyl-neuraminic acid.…”