Specificity of rat liver plasma membrane serine/threonine protein kinases and phosphatases over endogenous proteins.

The specificity of rat liver plasma membrane protein kinases and phosphatases was examined over endogenous substrates, using specific effectors of these enzymes. cAMP-dependent protein kinase was shown to phosphorylate the 77, 60 and 51 kDa phosphoproteins and type II casein kinase, a specific 24 kDa one. On the contrary, types 1 and 2A protein phosphatases seemed to have a broad specificity in plasma membranes. An analysis of the phosphoprotein pattern based on the endogenous substrates of plasma membrane enzymes was deduced from these and other results from our laboratory. The specificity of some enzymes might arise from the anchorage in plasma membrane which might restrict their activity to their immediate environment.

Stabilization of phospho-intermediates of rat liver plasma membrane alkaline phosphatase by uncompetitive inhibition. Relation with phosphate uptake into hepatocytes.

Rat liver plasma membrane alkaline phosphatase (ALP) phospho-intermediates, which have molecular masses of 151 and 135 kDa bands, were labelled at physiological pH with either (gamma-32P) ATP or 32Pi. This labeling was stabilized by a potent enzyme inhibitor, bromolevamisole (BL), and not by bromodexamisole (BD). BL augmented the rate and extent of autophosphorylation and slowed down the rate of autodephosphorylation of ALP. The phospho-intermediates labeling presented nearly the same kinetic behaviour with either (gamma-32P) ATP or 32Pi. In the presence of BL a marked decrease of the phosphorylation state of many proteins was observed in hepatocytes. BL also produced a decrease of the 32Pi uptake into hepatocytes and a decrease of the specific radioactivity of cellular ATP. BD had nearly the same effect as BL on protein phosphorylation and 32Pi uptake. These results argued against a direct involvement of ALP in Pi transport across hepatocyte plasma membrane.

Alkaline phosphatase activity at physiological pH: kinetic properties and biological significance.

The activity of rat liver alkaline phosphatase (ALP) was studied at physiological pH, using para-nitrophenyl phosphate (pNPP) as substrate. At this pH, the purified enzyme had optimal catalytic efficiency and its activity was maximal for the very low substrate concentrations. During thermal inactivation of rat liver plasma membranes activities, the ratio of the measured residual activities (pH 10.5/pH 7.5) varied, showing that ALP was not the only plasma membranes pNPP hydrolase. Indeed, the proportion of pNPP hydrolase activities attributable to ALP in plasma membranes at pH 7.5 was relatively low. Effectively, it was shown using bromolevamisole, a potent and specific inhibitor of ALP, that contrary to what it was previously reported, ALP was not the major pNPP hydrolase of liver plasma membrane.

Endogenous phosphorylation and dephosphorylation of rat liver plasma membrane proteins, suggesting a 18 kDa phosphoprotein as a potential substrate for alkaline phosphatase.

Purified rat liver plasma membranes were incubated for 0-60 min with [gamma-32P]ATP and analysis of 32P-labeled proteins by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the presence of two shifted kinetic phenomena. The use of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinases, allowed the identification of one as the endogenous protein phosphorylation. The other was shown to be the labeling of two phospho-intermediate forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1.], which have apparent molecular masses of 151 and 135 kDa. Bromolevamisole, a potent inhibitor of the enzyme, stabilized these phospho-intermediates, and consequent on this inhibition the labelling of a 18 kDa phosphoprotein was augmented. So, when alkaline phosphatase was studied in its native plasma membrane environment, a specificity of this enzyme over the endogenous phosphoproteins was established.

Enhancement of mitogen-induced lymphocyte proliferation by some inhibitors of alkaline phosphatase and diamine oxidase.

We selected various compounds [bromolevamisole, levamisole, cimetidine, L-homoarginine, 2,3,5,6-tetrahydroimidazo-(2,1-b)thiazole (IT), imidazole, theophylline] previously reported as inhibitors of alkaline phosphatase (ALP) and/or diamine oxidase (DAO) and studied their activity on concanavalin A (ConA)-induced mouse spleen cell lymphocyte proliferation. According to the Ki values, the decreasing order of potency for ALP inhibition was: bromolevamisole, levamisole, theophylline, cimetidine, IT, imidazole and L-homoarginine. The order of potency was different for DAO inhibition. Cimetidine was the most potent inhibitor of DAO, followed by bromolevamisole, levamisole, IT, imidazole and L-homoarginine. Theophylline had no inhibitory effect on DAO. We show that these compounds, except theophylline, enhance ConA-induced lymphocyte proliferation. Similarly, all the compounds except imidazole and theophylline, significantly inhibited ALP at concentrations which enhanced lymphocyte proliferation as measured by (3H)-thymidine uptake. DAO inhibition correlated with DNA synthesis only for IT and cimetidine. These observations suggest that ALP and DAO play a negative role in the proliferation process; however, the degree of enhancement of ConA-induced proliferation did not correlate strictly with the degree of ALP and DAO inhibition.

Comparative inhibition of human alkaline phosphatase and diamine oxidase by bromo-levamisole, cimetidine and various derivatives.

Analogues of bromo-levamisole and guanidine derivatives including cimetidine are examined in vitro in order to investigate their comparative inhibition, towards alkaline phosphatase (ALP) from human liver and diamine-oxidase (DAO) from human placenta. Bromo-levamisole, considered as a potent selective uncompetitive inhibitor of ALP (Ki, 2.8.10(-6) M at pH 10.5) is shown to be a noncompetitive inhibitor of DAO (Ki = 7.10(-4) M). According to the structure-inhibition relationship, the imidazole ring is important for ALP and DAO inhibition. The phenyl ring of bromo-levamisole is required for ALP inhibition but not for DAO inhibition, which is mediated mainly by aminoguanidine or guanidine groups. These results have allowed the selection of cimetidine, an H2-antagonist but also an immunomodulating compound, as inhibitor of these two enzymes. Cimetidine is an uncompetitive inhibitor of ALP (Ki = 3.2.10(-3) M at pH 10.5), and a good inhibitor of DAO (I50 = 3.8.10(-4) M). The Ki of ALP is commonly calculated at pH 10.5, but to study the role of the enzyme at the physiological pH, the inhibition has also been performed at pH 7.4. The Ki values are only slightly affected by this pH variation. So far several compounds, including levamisole, imidazole, theophylline and aminoguanidine are known to possess immunomodulating activities in vivo and/or in vitro and inhibit ALP and/or DAO. Therefore, it seems reasonable to assume that the inhibition of enzymes is involved in the immunomodulating effects of these drugs, when the ranges of active concentrations are similar for these properties.

Study of beta-glucosidase from Helix pomatia by active site-directed inhibitors.

This work describes the purification of a beta-glucosidase (beta-D-glucoside-glucohydrolase EC 3.2.1.21) from the digestive juice of Helix pomatia and the study of the enzyme's active site by using different reversible and irreversible inhibitors. The catalytic constants of arylglycosides and their pH-dependent variations have also been determined. The inhibition studies demonstrate that conduritol epoxides are irreversible inhibitors of beta-glucosidase from the digestive juice of H. pomatia, and that nojirimicin shows tight binding with glucosidase: the formation and dissociation of the enzyme-inhibitor complex (dissociation constant 1.1 mumol/1) required several minutes.

[The C cell system and experimental thyroid apudomas in the rat: the combined action of 2-acetylaminofluorene and chlorpromazine in inducing systemic medullary type tumors. An ultrastructural study]. / Le système des cellules C et les apudomes expérimentaux de la thyroïde chez le Rat: actions combinées du 2-acétylaminofluorène et de la chlorpromazine permettant l'obtention systématique de tumeurs de type médullaire. Etude ultrastructurale.

Previous chronic administration of chlorpromazine (CPZ) before the start of the 2-acetylaminofluorene (2-AAF) action and during the cancerogenic process systematically facilitate the growth of apudome-like medullary tumors inside the thyroid gland. Ultrastructural studies of normal C cells, adenomatous C cells and cancerous C cells derivated from the normal cellular type show various specific transformations of few intracytoplasmic organelles. In the Golgi apparatus, the path of main maturation of calcitonin-like granules seems the same in the normal and in the adenomatous cells. On the contrary, the amount of calcitonin-like granules falls in the tumoral extensive tissue even though the golgian activity turns towards a high abnormal proliferation of clathrin vesicles or coated vesicles. This new fact is directly related with numerous extensions and repairs of much damage of the neoplastic cell membranes. Finally, the large medullary tumors (showing many atypic mitosis in glandular neoplastic C cell-type) are deeply penetrated by extensive and irregular vegetative nervous fibers.

The use of inhibitors in the study of glycosidases.

The use of non-covalent as well as covalent inhibitors can be a useful tool to approach the mechanism of activity of glycosidases. An efficient method to determine the essential amino-acid groups directly or indirectly involved in the catalytic process is the use of active site directed irreversible inhibitors. Epoxide derivatives from conduritol B and conduritol C are the most important inhibitors in this group. The use of active site reversible inhibitors: cationic and basic glycosyl derivatives, glycals, glyconolactones, thioglycosides, is effective to study the different charges at the active site or the transition state during catalysis and also to detect conformational adaptability of an enzyme. Furthermore, inhibitors can be valuable tools to investigate various aspects of the physiological role of glycosidases.

[The main protein on the surface of the normal fibroblast (author's transl)]. / La protéine principale de la surface du fibroblaste normal.

In a general review, the authors discuss present knowledge on the main protein on the surface of the normal fibroblast, fibronectin. The peculiarities of this glycoprotein (localisation at the level of the cell surface, importance of thiol groups in the structure and expression of certain of its properties, reduction of the cells transformed by oncogenic viruses) are considered. The relationships which they may present with a plasma protein, cold insoluble globulin, are discussed together with the important role which they seem to play in the phenomena of cell adhesiveness.