Guido Van Dessel

The presence of phospholipase A2 in bovine adrenal medulla: Arguments for more than one type of phospholipase A2

Since phospholipase A2 (PLA2) is expected to play a role in the mechanism of exocytosis, the presence and subcellular localization of PLA2 in bovine adrenal medulla have been studied. The results of this study reveal that, although a large part of the PLA2 activity in chromaffin cells is due to a lysosomal PLA2, a cytoplasmic PLA2 is also present. This finding is supported by experiments in which the influence of pH, CaCl2 and NaCl on cytoplasmic PLA2 as well as the binding capacity to concanavalin A are investigated. According to these results the properties of a cytoplasmic PLA2 are clearly different from those reported by other authors for the lysosomal PLA2. For this reason, in chromaffin cells a PLA2 could be present which remains in the cytosol when the cell is in rest. Future experiments will have to prove whether this PLA2 becomes associated with the plasma membrane upon stimulation of the cell, thus mediating exocytosis.

Identification of prenylcysteine carboxymethyltransferase in bovine adrenal chromaffin cells.

Chromaffin cells from bovine adrenal medulla were examined for the presence of a specific prenylcysteine carboxymethyltransferase by using N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine as artificial substrates and a crude cell homogenate as the enzyme source. From Michaelis-Menten kinetics the following constants were calculated: K(m) 90 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-farnesyl-L-cysteine; K(m) 52 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-geranylgeranyl-L-cysteine. Both substrates were methylated to an optimal extent at the pH range 7. 4-8.0. Methylation activity increased linearly up to 20 min incubation time and was dose dependent up to at least 160 microg of protein. Sinefungin and S-adenosylhomocysteine both caused pronounced inhibition, as also to a lesser extent did farnesylthioacetic acid, deoxymethylthioadenosine and 3-deaza-adenosine. Effector studies showed that the methyltransferase activity varied depending on the concentration and chemical nature of the cations present. Monovalent cations were slightly stimulatory, while divalent metallic ions displayed diverging inhibitory effects. The inhibition by cations was validated by the stimulatory effect of the chelators EDTA and EGTA. Sulphydryl reagents inhibited methylation but to different degrees: Hg(2+)-ions: 100%, N-ethylmaleimide: 30%, dithiothreitol: 0% and mono-iodoacetate: 20%. Due to the hydrophobicity of the substrates dimethyl sulfoxide had to be included in the incubation mixture (<4%; still moderate inhibition at more elevated concentrations). The detergents tested affected the methyltransferase activity to a varying degree. The membrane bound character of the methyltransferase was confirmed.

Fluidity of Thyroid Plasma Membranes

The main function of the thyroid cell is the secretion of thyroid hormones (T4, thyroxine; T3, triiodothyronine). Thyroid hormones are first synthesized as part of thyroglobulin. Newly synthesized thyroglobulin is iodinated and vectorially transported to the apical region of the cell and released for storage in the lumen by exocytosis. When hormones are needed, thyroglobulin is removed from the luminal content by endocytosis and transferred to the lysosomes, where it is hydrolysed by cathepsin D liberating the hormones. Nonhormonal active iodotyrosines (3-monoiodotyrosine; 3,5-diiodotyrosine) are also formed. The hormones are released in the bloodstream and the iodotyrosine deiodinated. The iodide formed is partly reutilized by the cell (Taurog, 1978). This bidirectional transport of thyroglobulin is compartmented and implies concomitant transfer of membrane (Herzog, 1981). Every step in thyroid metabolism is activated by TSH (thyroid stimulating hormone, thyrotropin). It evokes intracellular metabolic responses by (1) recognition and binding to the cell surface, (2) transmission of information across the cell membrane, and (3) activation of internal metabolic pathways.

Characterization of dolichol kinase from bovine thyroid microsomes

Bovine thyroid microsomes are able to phosphorylate exogenous [1-3H]dolichol as well as endogenous dolichol. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [1-3H]dolichol to [1-3H]DMP as well as the formation of [32P]DMP from endogenous dolichol and [gamma-32P]CTP. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca2+- and Mn2+-ions. In accordance, EDTA was strongly inhibitory. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. The apparent Km-value for exogenous dolichol amounted to 4 microM. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [1-3H]dolichol depending on the source of CTP. With endogenous dolichol Km-values for CTP of 27.8 and 6.1 microM were calculated in respectively the absence and presence of 5 mM VO4(3-). Triton X-100 (0.15%) was necessary in the [1-3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [gamma-32P]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). The levels of the enzymatic activity could be doubled by the inclusion of 18-21 mM NaF [( 1-3H]dolichol kinase) as phosphatase inhibitor: VO4(3-) had practically no effect. In contrast with [gamma-32P]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO4(3-) while F- resulted into no appreciable effect.(ABSTRACT TRUNCATED AT 250 WORDS)

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose, hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue). Although the nuclei were more resistant to the isolation procedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gel electrophoresis.

Characterization and transcription of bovine thyroid chromatin

Abstract 1. 1. Thyroidal chromatin, isolated from bovine thyroid nuclei, has been characterized and the endogenous RNA polymerase activity was studied. 2. 2. The template activity of chromatin was investigated for exogenous homologous RNA polymerase I and II. 3. 3. The influence of chromosomal proteins on the transcription process was described.

Interaction of Cholera Toxin with its Receptor the Monosialoganglioside GM1: A Fluorescence Study

Cholera toxin (CT) is an enterotoxin secreted by Vibrio cholerae producing its pathological effects by increasing the c-AMP level in intestinal epithelial cells1,2. It is an oligomeric protein (Mr ~ 84,000) composed of two structural and functional distinct subunits CT A and CT B (Mr ~ 29,000 and 55,000 respectively). CT B contains five identical polypeptide chains (Mr = 11,600), most likely arranged in a ring-like pentameric configuration and CT A consists of two non-identical polypeptide chains A or a-chain (Mr = 23,000) and A2 or γ-chain (Mr = 5,500) linked by a single disulfide bridge (for reviews see refs. 1, 3–5). CT A is synthesized as a single polypeptide chain which is “nicked” between the two cysteine residues by an extracellular bacterial protease. During this proteolysis two serine residues are removed at the COOH terminus of A1 6. The first event in the action of CT on cells is the rapid, irreversible binding to receptors on the cell surface. It is generally accepted that the receptor for the toxin is the mono-sialoganglioside GM1 (for reviews see refs. 3,4,7).

Identification and characterization of a specific dolichylmonophosphate phosphatase activity in bovine thyroid microsomes

Bovine thyroid membranes are able to dephosphorylate exogenous [1-3H]DMP as well as endogenous prelabeled [32P]DMP. The kinetics, properties and specificity of the dolichylmonophosphatase activity have been studied by monitoring respectively the release of [1-3H]dolichol from [1-3H]DMP and the residual amount of [32P]DMP. The DMP-phosphatase activity is not linear with respect to time and exhibits a neutral pH optimum. There is only linearity in a narrow range of protein concentration when 0.25% Triton X-100 is included in the incubation mixture. Studying the enzymatic activity in function of protein concentration, the detergent requirement shows to be very critical. Triton X-100 is necessary for enzymatic activity with [1-3H]DMP (only 10% of enzymatic activity in the absence of detergent) although the detergent inhibits the hydrolysis of endogenous prelabeled [32P]DMP. Divalent cations are not essential for enzymatic activity, Ca2+-ions being even inhibitory. In accordance, EDTA (EGTA) is slightly stimulatory. The DMP-Pase activity is not influenced by the ionic strength of the incubation system and sulphydryl groups are not involved. NaF, VOS and VO4(3-) are strongly inhibitory. The inhibition by dolichol and PO3-4 can be explained as the result of product inhibition. An apparent Km-value of 2.5 X 10(-5) M is computed for [1-3H]DMP. Bacitracin inhibits DMP-phosphatase in contrast with other reports. Propylthiouracyl, cAMP, TSH and several other bio-effectors are without effect on the in vitro system. The specificity of the DMP-Pase activity is discussed, showing that the phosphatase is distinctly different from other phosphatases especially phosphatidic acid phosphohydrolase.