Reaction rate measurements of proteases and glycosidases with chromogenic methods

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.     

Effect of transforming growth factor beta (TGF-β) on ATP citrate lyase in isolated hepatocytes

Summary Transforming growth factor beta (TGF-) activates ATP citrate lyase in freshly isolated rat liver hepatocytes in a time dependent manner. Maximal stimulation of the enzyme occurred with less than thirty minutes of incubation of the cells with TGF-. The half maximal effect on the enzyme determined in hepatocytes incubated with TGF- for 10 min at 37°C was elicited by TGF- concentrations in the 10^–11 – 10^–12 M range. The potential role of TGF- stimulation of ATP citrate lyase activity in new membrane synthesis is discussed.     

Enzymatic and morphological response of the thymus to drugs in normal and zinc-deficient pregnant rats and their fetuses

In the thymus of normally fed pregnant rats the plasma membrane enzymes dipeptidyl peptidase IV (DPP IV) and alkaline phosphatase (alP) were found in cortical and medullary lymphocytes (thymocytes). Plasma membrane aminopeptidase A (APA) and adenosine monophosphate hydrolysing phosphatase (AMPP) were present in cortical reticular cells. In medullary reticular cells, aminopeptidase M (APM), gamma-glutamyl transferase (GGT), adenosine triphosphate (ATPP) and thiamine pyrophosphate (TPPP) cleaving phosphatases were detected. Medullary reticular cells did not contain APA. Lysosomal DPP I and II, acid phosphatase, acid beta-D-galactosidase, beta-D-N-acetyl-glucosaminidase, beta-D-glucuronidase and non-specific esterases occurred especially in macrophages at the corticomedullary junction. The 21-day-old fetal thymus showed a similar reaction pattern as the maternal organ except for APA which was absent before birth. After treatment of the pregnant rats with valproic acid (VPA), salicylic acid (SA), streptozotocin (ST) and retinoic acid (RA) APA showed an increase in activity in the thymic cortex. In addition, ST and RA induced AMPP, ATPP and TPPP activity in cortical reticular cells up to the same pattern as in medullary reticular cells. After ethanol (ET) administration severe damages occurred. The thymic cortex was free of DPP IV-positive lymphocytes; the medullary reticular cells showed reduced or no GGT and occasionally an increased APM activity. Dexamethasone (DEXA) given to normal or zinc-deficient rats produced the most severe lesions; thymocytes with DPP IV activity were completely absent in the cortex and medulla. In Zn-deficient pregnant rats similar alterations were observed as after ET.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of gentamicin-induced nephrotoxicity by Mg deficiency in non-pregnant rats. Morphological, enzyme histochemical and immunohistochemical studies

Non-pregnant rats fed an Mg-deficient diet showed some degenerated and calcified proximal tubules in the inner region of the medullary rays accompanied by reduced or absent enzyme activities. After gentamicin treatment some damaged convoluted proximal tubules occurred. Enzyme histochemistry revealed decreased activities for brush border and lysosomal hydrolases; using immunohistochemistry lesions were detectable for the cytoskeletal proteins keratin and vimentin. Administration of gentamicin to Mg-deficient rats led to a further decrease of hydrolase activities in obviously intact proximal tubules and drastic structural and enzymatic defects as well as alterations of the cytoskeletal proteins in the convoluted and straight segments of other proximal tubules and to a lesser degree also in glomeruli and further portions of the tubular apparatus including the collecting ducts.     

Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features

Charybdotoxin (CTX), a 37 amino acid protein isolated from the venom of L. quinquestriatus, is a high-affinity blocker of various Ca2(+)-activated K+ channels. CTX also blocks Drosophila Shaker (Sh) clone H4 transient K+ currents expressed in Xenopus oocytes with similar affinity (Kd = 3.6 nM). CTX blocks both the open and the closed states of Sh channels with no apparent change in gating behavior. In addition, the block is enhanced as the ionic strength is lowered. These properties are identical to those of CTX block of Ca(+)-activated K+ channels, and these results suggest that the external pore openings of these two functionally dissimilar K+ channels may share common structural features.     

Light-microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes

Summary New lanthanide methods for the histochemical detection of non-specific alkaline phosphatase in the light microscope are described and compared with already existing techniques for the light microscopical demonstration of this enzyme. To avoid formation of insoluble lanthanide hydroxide at alkaline pH citrate complexes with the capture ions cerium, lanthanum and didymium were used. A molar ratio of 11 mM citrate/14 mM capture reagent is proposed. For preincubated sections, pretreatment in chloroform-acetone and fixation in glutaraldehyde, for non-preincubated sections fixation in glutaraldehyde yielded the best results. 4-Methylumbelliferyl and 5-Br-4-Cl-3-indoxyl phosphate were found to be the most suitable substrates. For routine purposes 4-nitrophenyl, 1-naphthyl, 2-naphthyl and 2-glycerophosphate were also sufficient; naphthol AS phosphates were inferior but still suitable. After incubation for 5–60 min at 37° C lanthanide phosphate was converted into lead phosphate which was visualized as lead sulfide. At pH 9.2–9.5 enzyme activity was demonstrated at many sites such as intestinal, uterine, placental, renal and epididymal microvillous zones, plasma membranes of arterial, sinus and capillary endothelial cells, vaginal and urethral epithelium, smooth muscle cells, myoepithelial cells as well as excretory duct cells of salivary and lacrimal glands and in secretory granules of laryngeal glands. In comparison with Gomori's calcium, Mayahara's lead, Burstone's and Pearse's azo-coupling, McGadey's tetrazolium salt and Gossrau's azoindoxyl coupling technique the lanthanide methods detected alkaline phosphatase activities at identical or additional sites depending on the respective procedure. However, in contrast to the other methods especially the cerium citrate procedure yielded a more precisely localized and more stable reaction product, can be used with all available alkaline phosphatase substrates including those up till now less suitable or unsuitable for light microscopic alkaline phosphatase histochemistry.     

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure

Summary New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca-and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H₂O₂ which decomposes at 55°–60° C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H₂O₂ mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H₂O₂-DAB and especially the H₂O₂-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.     

Membrane protein phosphorylation during stomatocyte-echinocyte transformation of human erythrocytes

The normal, discoid shape of red blood cells represents an equilibrium between two opposing factors, i.e., stomatocytic and echinocytic transformations. Most stomatocytic agents were found to be inhibitors of calmodulin, a regulator of the phosphorylation of membrane proteins. We determined whether red cell shape transformations could be caused by changes in phosphorylation of membrane proteins, specifically the cAMP-dependent phosphorylation of ankyrin and band 4.1. Red blood cells were incubated with 32P and 100 microM chlorpromazine (stomatocytic transformation) or 30 mM sodium salicylate (echinocytic transformation) for various time intervals. Ghost membrane proteins were examined by polyacrylamide gel electrophoresis and autoradiography. Spectrin (beta-chain), ankyrin, band 3, band 4.1 and 4.9 were phosphorylated. No change was found in the degree and pattern of phosphorylation after stomatocytic transformation. Salicylate caused a reversible inhibition of transmembranous phosphate transport in both directions. The results indicate that the stomatocytic transformation induced by chlorpromazine and the echinocytic transformation induced by salicylate do not involve a change in phosphorylation, but that the echinocytic transformation induced by salicylate is associated with an inhibition of transmembranous transport of phosphate. Studies with salicylate suggest that the phosphorylation sites of band 3 are found mainly on the endofacial side of the membrane.     

Protease cytochemistry in the murine rodent,guinea-pig and marmoset placenta

Summary Plasma membrane and lysosomal proteases, -glutamyl transferase and extracellular matrix proteases were investigated by qualitative cytochemical means in the mature placenta of mice, rats, guinea-pigs and marmosets. These studies revealed similarities, which concerned, primarily the lysosomal proteases in different structures of the placenta and all proteases and -glutamyl transferases in the zone of placental shedding. However, species differences predominated. They were observed especially for aminopeptidase A and M, dipeptidyl peptidase IV and -glutamyl transferase in the plasma membranes and extracellular matrix of the placental barrier and decidual cells of all species and the cells of the basal zone in rats and mice. Plasma membrane and extracellular matrix proteases in other parts of the placenta, e.g. the placenta stem of guinea-pigs and basel plate, amniotic and chorionic plate of marmosets occurred only in these species. Elastase substrates hydrolysing endopeptidase I and kallikrein-, thrombin-, plasmin-, plasminogen- and cathepsin B substrates hydrolysing endopeptidase II were not observed in any of these species. A general comparison of the species revealed similarities for the mouse, rat and guinea-pig placental barrier, but not for that of marmosets. The proteases of this zone in the marmoset placenta are more similar to the human situation, but do not correspond to it completely.In honour of Prof. P. van DuijnSupported by the BMFT (Project CMT 35) and Sfb 174In part presented at a Symposion on Progress in General, Applied and Diagnostic Histochemistry (Modra, Czechoslovakia on 12–15 April, 1984; abstracts published in Histochem J 17, No 5, 1985)