Chicken liver fatty acid synthetase dimer: Evidence of asymmetrical structure from iodoacetamide inhibition studies

The condensing component of chicken liver fatty acid synthetase is inhibited by a sulfhydryl reagent, iodoacetamide, with a second-order rate constant of 0.23 M^–1 sec^–1 at pH 7.0 and 0. Complete inactivation requires the modification of approximately 8-SH groups per dimer of the enzyme. Quantitation of the extent of inactivation in the presence of i mM acetyl CoA (which completely protects the enzyme against inactivation) and in its absence shows that complete inactivation results from the binding of approximately 1.1 tool of carboxamidomethyl residues per dimer. These data are consistent with the proposed functional asymmetry of the enzyme.     

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

The study, addressed to understand whether or not human platelets possess a unique thiol-oxidase whose activity could be modulated by signalling pathway initiated upon the activation of Receptor-C_k revealed the existence of disulphide-dependent oxidation within these cells and this phenomenon was regulated by Receptor-C_k-dependent generation of second messengers especially phosphatidic acid (PA); cAMP and cGMP. Purification of this activity revealed the existence of 47 kDa protein having thiol-oxidase activity. Keeping in view these results we propose that the existence of this novel 47 kDa Thiol-oxidase within human platelets may provide a crucial switch for the regulation of Receptor-C_k-dependent mevalonate pathway in human platelets.     

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor - alpha (TNF-) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF- inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF- at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF- inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.     

Fatty acid synthetase complex: Selective inactivation by phenylmethylsulphonyl fluoride

Reaction of pigeon and rat liver fatty acid synthetases with phenylmethylsulphonyl fluoride at pH 7.0 results in rapid and complete loss of activity for fatty acid synthesis. Acetyl and malonyl transacylation, two reductions, dehydration and condensation-CO₂ exchange reactions are not appreciably altered in the modified enzyme. However, the deacylation of palmityl CoA is completely inhibited. Complete inactivation results in the incorporation of about 1.9 moles of ¹⁴C-phenylmethylsulphonyl groups/mole of the enzyme complex. These results suggest that either two moles of a fatty acyl deacylase or two deacylases with different fatty acyl chain length specificities may be functional in the enzyme complex.     

Irreversible inactivation of chicken liver fatty acid synthetase by its substrates acetyl and malonyl CoA: Effect of temperature and NADP+ on fatty acid and triacetic acid lactone synthesis

Chicken liver fatty acid synthetase is irreversibly inactivated by malonyl CoA and by acetyl and malonyl CoA. Two active forms of the enzyme existing above and below 11.5° are inactivated at different rates. Activities for fatty acid and triacetic acid lactone synthesis are lost at about the same rate and NADP⁺ protects the enzyme against inactivation. Inactivation results from the enhanced covalent binding of malonyl groups in addition to those required for fatty acid synthesis.