Isolation and synthesis of two new flavones from Conoclinium coelestinum

Two new flavones, 3′,4′-methylenedioxy-5,6,7,8,5′-pentamethoxyflavone and 5,7,4′-trihydroxy-6,3′,5′-trimethoxyflavone, have been isolated from Conoclinium coelestinum. Final structure proof was accomplished by synthesis.     

Formation of γ-glutamyl-ϵ-lysine bridges between membrane proteins by a Ca2+-regulated enzyme in intact erythrocytes

A rise in the intracellular concentration of Ca²⁺-ions in human erythrocytes causes the formation of high-molecular-weight membrane protein polymers, cross-linked by γ-glutamyl-?-lysine side chain bridges. Cross-linking involves proteins at the cytoplasmic side of the membrane (band 4.1, spectrin, and band 3 materials) and the reaction is catalyzed by the intrinsic transglutaminase. This enzyme is regulated by Ca²⁺-ions and it exists in a latent form in normal cells. The protein polymer, isolated from the membranes of Ca²⁺-loaded intact human red cells, is heterogeneous in size and may contain as many as 6 moles of γ-glutamyl-?-lysine cross-links per 100,000 gm of protein. Synthetic compounds, which either compete against the ?-lysine cross-linking functionalities of the protein substrates (eg, histamine, aminoacetonitrile, cystamine) or directly inactivate the transamidase (eg, cystamine), inhibit the membrane polymerization reaction in intact human erythrocytes. They also interfere with the Ca²⁺-induced irreversible shape change from discocyte to echinocyte and inhibit the irreversible loss of membrane deformability. Thus, the transamidase-catalyzed production of γ-glutamyl-?-lysine cross-links in the membrane may be a common denominator in these cellular manifestations.     

Cross-linked polymers in the red cell membranes of a patient with Hb-Koln disease

Membranes isolated from the red cells of a Hb-Koln patient were shown to contain appreciable amounts of N epsilon-(gamma-glutamyl)lysine, the characteristic product of the post-translational cross-linking of proteins by transglutaminase. Such an apparently irreversible fusion of membrane proteins may contribute to the membrane defect in Hb-Koln disease.     

Enzymatic basis for the Ca2+-induced cross-linking of membrane proteins in intact human erythrocytes.

The accumulation of Ca2+ ions in intact human erythrocytes leads to the production of membrane protein polymers larger than spectrin. The polymer has a heterogeneous size distribution and is rich in gamma-glutamyl-epsilon-lysine cross-links. Isolation of this isodipeptide, in amounts as high as 6 mol/10(5) g of protein, confirms the idea [Lorand L., Weissmann, L.B., Epel, D.L., and Bruner-Lorand, J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 4479] that the Ca2+-induced membrane protein polymerization is mediated by transglutaminase. Formation of the polymer in the intact cells is inhibited by the addition of small, water-soluble primary amines. Inasmuch as these amines are known to prevent the Ca2+-dependent loss of deformability of the membrane, it is suggested that transglutaminase-catalyzed cross-linking may be a biochemical cause of irreversible membrane stiffening.     

Relations between fatty acids and oestrogen binding properties of pure rat alpha 1-foetoprotein.

A delipidation procedure based on treatment with charcoal at pH 3 has been applied to highly purified rat alpha 1-foetoprotein preparations. The oestrogen binding properties of the delipidated proteins have been studied with an equilibrium dialysis technique, and compared with the properties of the untreated foetal protein, as well as those of preparations reconstituted from the defatted alpha 1-foetoprotein and the removed lipids. An important increase has been evidenced for the binding levels of oestrone, oestradiol-17 beta and diethylstilboestrol by the delipidated alpha 1-foetoprotein. A reversal of this effect has been obtained by incubating the delipidated protein either with the lipids extracted from the purified alpha 1-foetoprotein or with a potent competitor of the rat alpha 1-foetoprotein-oestrogen interaction, designated as 'L', previously demonstrated and isolated from whole rat sera, and tentatively characterized as a mixture of fatty acids. Scatchard analysis of the oestrone and oestradiol-17 beta binding parameters show that the enhanced fixation of the hormones after defatting is primarily due to a two-fold increase of the apparent number of binding sites/mol alpha 1-foetoprotein. The results are interpreted in terms of the probable, at least partial, identity between the lipids closely associated with the pure alpha 1-foetoprotein and the fatty acid mixture 'L' isolated from whole sera. The possible biological role of complex interplay between oestrophilic alpha 1-foetoproteins, phenolsteroids and fatty acids in the control of oestrogen levels during development is discussed briefly.     

The microheterogeneity of rat TBG

Isoelectric focusing (IEF) of native sera from immature or adult rats and of purified or partially purified rat serum thyroid hormone-binding proteins, demonstrates that rat TBG is a microheterogeneous protein. Autoradiography and radioactivity scans of the IEF plates show that it consists of at least four main isoforms, with bands at pH 4.35, 4.45, 4.5 and 4.55. This pattern is remarkably similar to that reported for human TBG. This is the first demonstration of the polymorphism of this recently discovered major binding protein of the rat.