Kinetic studies on the action of Mucor pusillus, Mucor miehei acid proteases and chymosins A and B on a synthetic chromophoric hexapeptide.

The action of two milk-clotting fungal proteases from Mucos pusillus and Mucor miehei and of chymosins A and B on the hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, and on kappa-casein were studied. The effects of pH and temperature on the initial rates of hydrolysis of the hexapeptide were examined. Crystalline chymosin and M. pusillus protease exhibited optimal activities around 49 and 55 degrees C, respectively, whereas the optimum temperature for M. miehei protease is higher than 63 degrees C. The optimum pH was about 4.7 for both fungal proteases whereas chymosin A and chymosin B exhibited optimal activities around 4.2 and 3.7, respectively. Kinetic parameters were then determined under optimal conditions and/or at pH 4.7. Fungal proteases had kcat/Km ratios that were similar to each other and that were significantly greater than the ratios obtained for the chymosins. Nevertheless, chymosins had much greater clotting activities towards kappa-casein relative to their proteolytic activities towards the synthetic peptide.

Effect of microtubule network disturbance by nocodazole and docetaxel (Taxotere) on protein secretion in rat extraorbital lacrimal and parotid glands.

The role of microtubules in the exocrine secretory process is not yet well established. Contradictory effects of anti-microtubule drugs on intracellular transit and protein secretion have been reported. In this work we used microscopic techniques and pulse-chase experiments to compare the involvement of microtubules in the regulated secretory process of two rat exocrine glands: parotid and extraorbital lacrimal glands. In our experiments microtubules were either disrupted by nocodazole or stabilized by a taxoid, docetaxel (Taxotere). We show that the effect of nocodazole and docetaxel on the release of newly synthesized proteins is radically different in the two tissues; in parotid gland they only weakly affect protein release, triggered by stimulation of either muscarinic or beta-adrenergic receptors, but in lacrimal gland, they strongly inhibit protein secretion. This effect or lack of effect of the drug is independent of the signal transduction pathways involved by the different secretagogues used to trigger exocytosis. Furthermore, in lacrimal glands, studies on protein galactosylation (which occurs in the trans-Golgi compartment) indicate that postgalactosylation events are more sensitive to both drugs than pregalactosylation events. On the other hand, we show that the effect of nocodazole and docetaxel on the microtubule network is comparable on the two tissues. Finally, in lacrimal cells, we observed a scattering of the Golgi apparatus concomitant with the disruption of microtubules by nocodazole. We conclude from this study that microtubule network integrity is essential for protein secretion in lacrimal glands but not in parotid glands. This result implies that for the same physiological function, i.e. protein secretion, different mechanisms may be involved.

DNA synthesis and microtubule assembly-related events in fertilized Paracentrotus lividus eggs: reversible inhibition by 10 mM procaine.

This report describes the effects of 10 mM procaine on microtubule assembly and on DNA synthesis, as followed by [3H]colchicine binding assays and [3H]thymidine incorporation respectively, in fertilized Paracentrotus lividus eggs. In the absence of microtubule assembly inhibitors, about 25% of the total egg tubulin is submitted to two cycles of polymerization prior to the first cell division, this polymerization process precedes DNA synthesis. If the zygotes are treated with 10 mM procaine in the course of the cell cycle, tubulin polymerization is inhibited or microtubules are disassembled. DNA synthesis is inhibited when procaine treatment is performed 10 min, before the initiation of the S-period. However, when the drug is applied in the course of this synthetic period, the process is normally accomplished, but the next S-period becomes inhibited. Moreover, procaine treatment increases the cytoplasmic pH of the fertilized eggs by about 0.6 to 0.8 pH units. This pH increase precedes microtubule disassembly and inhibition of DNA synthesis. Washing out the drug induces a decrease of the intracellular pH which returns to about the same value as that of the fertilized egg controls. This pH change is then followed by the reinitiation of microtubule assembly, DNA synthesis and cell division. Our results show that the inhibition of both tubulin polymerization and DNA synthesis in fertilized eggs treated with 10 mM procaine, appears to be related to the drug-induced increase in cytoplasmic pH.

Isolation of a 50 kDa polypeptide from the detergent-resistant unfertilized sea urchin egg cytomatrix and evidence for its change in organization during mitosis.

In this report, we describe the isolation of a 50 kDa polypeptide from the detergent-resistant cytomatrix of unfertilized sea urchin egg. This polypeptide shares with the intermediate filaments the property of insolubility in high ionic strength buffer solution. However, it does not cross-react with anti-vimentin and anti-cytokeratin antibodies. Studies performed by indirect immunofluorescence microscopy with an immunospecific serum raised against this polypeptide show that during the first cell cycle the polypeptide exhibits similar configuration changes as those described for tubulin. Using immunocytochemical light and electron microscopy, we present evidence indicating that this 50 kDa polypeptide is a constituent of the isolated mitotic apparatus; it is mainly located on patches of microfibrillar material found close to the microtubules. The 50 kDa polypeptide is not extracted from taxol-assembled microtubules by the 0.6 M NaCl treatment. However, the difference in solubility between this protein and the previously studied microtubule-associated proteins does not preclude the possibility of the 50 kDa polypeptide on being a "microtubule-associated protein". The possible significance of this novel cytoskeletal component is discussed.

[Prolactin internalisation by the epithelial mammary cell: effects of lysosomotropic agents and transglutaminase inhibitors]. / Endocytose de la prolactine dans la cellule épithéliale mammaire: effets des agents lysosomotropes, et des inhibiteurs de la transglutaminase.

Prolactin endocytosis was studied by electron microscopy with 125I-prolactin 125I-hGH (human growth hormone) and prolactin-ferritin. Endocytosis and intracellular transit of the labelled hormone proceeded identically in epithelial cells isolated from the mammary glands of pseudopregnant rabbits and in surviving fragments from mammary glands of lactating rabbits. After binding of the hormone to its receptor, the labelled material was rapidly detectable in vesicles showing an homogeneous aspect; 15 min later part of the labelled material was still localized within the same kind of vesicles, but in addition it appeared to have migrated into microvesicles of the Golgi region and into vesicles of heterogeneous aspect tentatively identified with lysosomes. Endocytosis of bovine serum albumin, labelled with ferritin followed the same intracellular pathway. Native ferritin accumulated in vesicles of various sizes, but seemed excluded from the microvesicles of the Golgi zone. In the presence of lysosomotropic agents labelled prolactin accumulated in cytoplasmic vesicles. In the presence of dansylcadaverine, endocytosis of the labelled material proceeded unimpaired. Conversely, in the presence of bacitracin, the internalisation of labelled prolactin seemed to be reduced. These observations show that the endocytosis of the hormone/receptor complex is linked to membrane movements, which eventually lead to its location within both the Golgi apparatus and the lysosomes.

Cell-free synthesis, proteolytic processing, core glycosylation, and amino terminal sequence of rabbit pre-alpha-lactalbumin.

Two different forms of alpha-lactalbumin were isolated from rabbit milk and partially characterized. The major and the minor species had apparent molecular weights of 18000 and 14000, respectively, according to their electrophoretic mobilities on SDS polyacrylamide gels. Analyses of their amino acid compositions and amino-and carboxy-terminal sequences did not reveal any difference, but sugar analysis showed the occurrence of carbohydrates in the major species. Rabbit alpha-lactalbumin was synthesized in a cell-free translation system as a precursor with an amino terminal extension of 19 amino acid residues whose primary structure is rather different from those of its ovine and porcine counterparts, in contrast with the extensive similarity so far observed between the known signals of homologous milk proteins. When mammary microsomal membranes were added during translation, the preprotein was converted to authentic alpha-lactalbumin, as demonstrated by amino terminal sequence analyses. However, one of the two processed forms migrated more slowly than pre-alpha-lactalbumin on SDS polyacrylamide gels and this was related to the occurrence of carbohydrates: only the "slower moving" polypeptide was specifically adsorbed on concanavalin A Sepharose and its electrophoretic mobility was enhanced after treatment with endoglycosidase H, an enzyme known to remove clustered mannosyl residues linked to di-N-acetylchitobiose. It was also observed that the rate of translocation of alpha-lactalbumin across the microsomal membrane was lower than that of beta-casein.

Amino terminal sequence, processing, and biological activity of porcine pre-alpha-lactalbumin.

Polyadenylated RNAs isolated from bound polysomes of a lactating sow's mammary gland, were translated in a cell-free system and in vitro synthesized alpha-lactalbumin was immunoprecipitated and radiosequenced. The translation product was found to contain an amino terminal extension of 19 amino acid residues, very similar to its ovine counterpart, that was selectively removed when translation was carried out in the presence of rabbit mammary microsomal membranes. Assays of porcine pre-alpha-lactalbumin for activity on galactosyltransferase showed that the preprotein can also interact with and modify the specificity of the enzyme, as indicated by de novo synthesis of lactose.

Association between microtubules and Golgi vesicles isolated from rat parotid glands.

We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 micrograms of microtubule protein bound to 500 micrograms of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 micrograms/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10-20% of the total tubulin content of the parotid cell.

Are microtubules essential for the secretory process in rat parotid gland?

The role of microtubules in the exocrine secretory process is not yet well established, and their disruption by anti-microtubule drugs leads to variable effects on intracellular transit and protein secretion. We investigated the involvement of microtubules in the regulated secretory process of rat parotid glands using microscopic techniques and pulse-chase experiments. We showed that 10 microM colchicine or nocodazole destroys the microtubule network in parotid acinar cells but only weakly reduces the release of newly synthesized proteins. The half-effect was obtained with 0.22 microM colchicine. Moreover, this small reduction was found to be independent of the nature of the drug (colchicine, colcemid, or nocodazole) and of the nature of the stimulation (beta-adrenergic or cholinergic pathways). Using nocodazole, we have been able to determine that the steps affected by the drug are very early events in the secretory pathway. Finally, we showed by kinetic analysis that microtubule disruption slows protein release only moderately but does not reduce the total amount of secreted protein. We conclude from this study that microtubule integrity is not essential for protein secretion in rat parotid gland.

Cytoskeleton of the unfertilized sea urchin egg.

Unfertilized Paracentrotus lividus egg cytoskeleton is prepared by mild, nonionic detergent extraction at 4 degrees C in buffer systems containing either 2-methyl-2,4-pentanediol (hexylene glycol) or glycerol. These extractions allow the isolation of cytomatrices that maintain the egg form and are 70-80 micron in diameter. DNase inhibition assays show that actin is in polymerized form in these cytomatrices. Ultrastructural observations reveal that the cytoskeletons are made up essentially of 2 categories of filaments, 7-8-nm and 2-4-nm in diameter, respectively. After heavy meromyosin labelling, short, radiating actin filaments are seen in the cortical region, while longer actin filaments are found in the internal region of these cytomatrices. The 2-4-nm filaments of still unknown biochemical nature are organized in a meshwork. In contrast to results found with fertilized eggs, bundles of actin filaments and microtubules are absent; 8-13-nm filaments are not detected.

Detoxification mechanism in the rat extraorbital lacrimal gland: conjugation of brefeldin A to glutathione.

In this work the existence of a glutathione based detoxification system in rat lacrimal glands is reported. We showed that brefeldin A, a drug used as a tool for the study of intracellular trafficking mechanisms, was inactivated by metabolization and converted into two derivatives. We purified them by high performance liquid chromatography and determined, by mass spectroscopy, that they correspond to glutathione and cysteine derivatives of BFA. The determination of the respective amounts of these derivatives in the medium and the tissue in different experimental conditions, revealed that glutathione-BFA is formed in the tissue, excreted from the cells, cleaved by gamma-glutamyl transpeptidase and finally converted to cysteine-BFA.

Recovery of protein secretion after brefeldin A treatment of rat lacrimal glands: effect of cAMP.

In exocrine cells, the discharge of secretory granule contents in response to extracellular stimuli has been widely documented. However, few data are available concerning the effect of these stimuli on the steps of the secretory pathway preceding protein exocytosis. To obtain more data on this subject, we used brefeldin A (BFA) to perturb intracellular protein transit. When, after exposure of the lacrimal gland lobules to 10 microM BFA, which led to a complete dismantling of the Golgi apparatus and fully inhibited the secretion of newly synthesized proteins, the drug concentration was lowered to 100 nM, a restoration of protein secretion was observed in a secretagogue-dependent manner. Secretagogues increasing the adenosine 3',5'-cyclic monophosphate (cAMP) level facilitated the recovery of protein secretion and Golgi apparatus restructuring, whereas other secretagogues, involving the calcium pathway, did not. Furthermore, the cAMP effect was prevented by H-89, a specific protein kinase A inhibitor. These effects of cAMP are due to neither BFA degradation nor BFA excretion from the cells. We conclude from these results that in rat lacrimal glands the recovery from the dramatic damage caused by BFA is promoted by a cAMP-dependent mechanism and further suggest a role of cAMP in the regulation of the Golgi structure and/or function.

Tubulin dynamics during the cytoplasmic cohesiveness cycle in artificially activated sea urchin eggs.

Sedimentation studies and [3H]colchicine-binding assays have demonstrated a relationship between the cytoplasmic cohesiveness cycles and the changes in tubulin organization in Paracentrotus lividus eggs activated by 2.5 mM procaine. The same amount of tubulin (20-25% of the total egg tubulin) is involved in these cyclic process and appears to undergo polymerization and depolymerization cycles. Electron microscopy studies reveal that the microtubules formed during these cytoplasmic cohesiveness cycles are under a particulate form which is sedimentable at low speed. Activation experiments carried out in the presence of cytochalasin B (CB) show that the increase in the cytoplasmic cohesiveness is highly reduced while tubulin polymerization and depolymerization cycles and pronuclear centration are not affected. Although tubulin or actin polymerization can be independently triggered in procaine-activated eggs, the increase in cytoplasmic cohesiveness requires the polymerization of both proteins. However, the cytoplasmic cohesiveness cycles appear to be regulated by tubulin polymerization and depolymerization cycles.

PKA inhibitor, H-89, affects the intracellular transit of regulated secretory proteins in rat lacrimal glands.

We tested the effect of H-89, a protein kinase A (PKA) inhibitor, on the intracellular transit of the regulated secretory proteins in rat lacrimal glands. We show that H-89, by itself, induces the secretion of newly synthesized proteins trafficking in its presence but not of proteins already stored in the mature secretory granules. This secretion does not depend on the presence of extracellular Ca2+. The proteins released are identical to those secreted after cholinergic stimulation or under the action of the ionophore A-23187, but the secretion level is approximately 40% lower. The effect of H-89 seems to be due to PKA inhibition because other protein kinase inhibitors (calphostin C, chelerythrine, H-85) do not induce secretion. We further show that H-89 does not modify the rate of glycoprotein galactosylation but induces the secretion of newly galactosylated glycoproteins. Finally, we used a "20 degrees C block" procedure to show that H-89 affects a trans-Golgi network (TGN) or post-TGN step of the secretory pathway. Our results demonstrate that, in lacrimal cells, H-89 affects the intracellular trafficking of secretory proteins, suggesting a role for PKA in this process.

[Study of a lysis medium stabilizing microfilaments and microtubules in vitro and in vivo]. / Etude d'un milieu de lyse stabilisant les microfilaments et les microtubules in vitro et in vivo.

Determination of experimental conditions which allow the evaluation of the variations in the ratio of non polymerized and polymerized forms of actin and tubulin during the reorganization of the cytoskeletal cell system is of most valuable importance. In order to prepare cell homogenates which would reflect the in vivo situation, we tested in vitro a lysis medium which stabilized both microfilaments and microtubules, which were determined by DNase inhibition assays and colchicine binding assays respectively. This lysis medium containing 10 mM potassium phosphate, 1mM magnesium chloride, 5 mM EGTA, 1 M hexylene glycol, 1% Triton X-100, pH 6.4, used at 4 degrees C a) diffused rapidly into the cells; b) did not denature actin and tubulin; c) did not displace the equilibrium between non polymerized and polymerized forms of actin and tubulin, allowing biochemical assays on cell homogenates; d) blocked the evolution of the cytoskeletal system and permitted structural studies; e) and allowed the decoration of microfilaments by heavy meromyosin.

Dual effect of procaine in sea urchin eggs. Inducer and inhibitor of microtubule assembly.

An increase in the amount of cytoplasmic filamentous structures (cytoplasmic matrix and aster) which were recovered after hexylene glycol/Triton X-100 treatment of sea urchin eggs (Paracentrotus lividus) activated by 0.2-2.5 mM procaine was observed. At higher activator concentrations, an opposite effect was observed and formation of these cytoplasmic structures was inhibited in the presence of 10 mM procaine. This inhibitory effect was reversed by diluting the drug in the incubation medium. DNase I inhibition assays on egg homogenates which were performed at different time points of the activation process, show that the same amount of actin was induced to polymerize in eggs activated either by 2.5 or 10 mM procaine. However, colchicine-binding assays on the 100 000 g particulate fractions of these homogenates show that in eggs activated by 10 mM procaine, in contrast to those activated by 2.5 mM, tubulin polymerization was inhibited and microtubules were disassembled. These results show that the dual effect of procaine in the organization of the egg cytoskeleton appears to be related to its effect on the state of tubulin.