Fluorescence studies of the aged erythrocyte membranes

The most important purpose of this research is to characterize by means of fluorescence polarization the structural and functional changes which occur in the membrane of the human erythrocytes during aging process. Our results provide evidence of a significant increase of membrane fluidity in the deep lipid core and in the lipid/protein boundary, in the aged erythrocytes. These features are associated with a rigidity of the membrane surface, as revealed by the anisotropy increase of a specific probe suitable for monitoring the membrane protein behaviour. These modifications could be considered as one of the mechanisms which contribute to alter erythrocyte rheological properties sufficiently to be recognised and removed within circulation.     

Fluorescence studies on erythrocyte membrane isolated fromPlasmodium berghei infected mice

The erythrocyte host cell plays a key role in the well defined developmental stages of the malarial parasite growth and propogation in the erythrocyte cycle of malaria. The host cell serves the parasites by supplying metabolites and removing the catabolites produced by the obligatory parasites. It has been observed that the plasma membrane of the infected cells show a substantially higher fluidity probably due to the depletion of cholesterol content from the host cell. The protein component of the membrane is also modulated due to the insertion of new polypeptides of the parasitic origin, which confers upon it new antigenic properties. We have studied the membrane fraction isolated from mice erythrocytes infected withPlasmodium berghei using fluorescent probes like DPH, ANS and series of fluorenyl fatty acids, which permit depth dependent analysis of membrane. We have observed that there is a marked difference in the fluorescence emission wavelength maximum, the dissociation constant K_d of ANS when bound to normal and infected erythrocytes, though relatively small differences are observed in the fluorescence polarisation values of the two cell types. The fluorenyl fatty acids also show the differences when bound to normal and infected erythrocytes, indicating that either they are in a different environment or they have differing binding properties to the two cell types.Abbreviations DPH 1,6-Diphenyl-1,3,5-Hexatriene - ANS 8-Anilino-napthalene Sulfonic Acid - C2A-FL 2-Fluorenyl-acetic Acid - C4A-FL 2-Fluorenyl-butyric Acid - C6A-FL 2-Fluorenyl-hexanoic Acid - C8A-FL 2-Fluorenyl-octanoic Acid     

Fluorescence studies on calmodulin binding to erythrocyte Ca2(+)-ATPase in different oligomerization states

The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2(+)-ATPase. Previous studies have shown that the Ca2(+)-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2(+)-dependent manner, with a approximately 50% fluorescence enhancement. These findings confirm our previous observation that enzyme oligomers retain their ability to bind calmodulin, even though they are fully activated in the absence of calmodulin. The Ca2+ dependence of MIANS-CaM binding to monomeric Ca2(+)-ATPase is of higher affinity (K 1/2 = 0.09 microM Ca2+) and less cooperative (nH = 1.1) than the Ca2+ dependence of enzyme activation by MIANS-CaM (K 1/2 = 0.26 microM Ca2+, nH = 2.8). These Ca2+ dependences and the order of events, in which calmodulin binding precedes enzyme activation, demonstrate that calmodulin indeed could be a physiological activator of the monomeric enzyme. The calcium dependence of calmodulin binding to oligomeric Ca2(+)-ATPase occurs at even lower levels of Ca2+ (K 1/2 = 0.04 microM Ca2+), in a highly cooperative fashion (nH = 2.3), and essentially in parallel with enzyme activation (K 1/2 = 0.05 microM Ca2+, nH = 2.9). The observed differences between monomers and oligomers suggest that the oligomerized Ca2(+)-ATPase is in a conformation necessary for efficient, cooperative calcium binding at nanomolar Ca2+, which the monomeric enzyme acquires only upon interaction with calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence studies on concanavalin-A

Using the lectin-concanavalin-A, the tryptophan fluorescence as a function of pH was studied. The pH dependent, fluorescence intensity changes were significantly higher when excited at 305 nm, than when irradiated at 280nm. Only one tryptophanyl per monomer of concanavalin-A was available for oxidation by N-bromosuccinimide in the dimeric form at pH 4·9; no tryptophanyl could be oxidised in the demetallised dimer (pH 3·0) and native tetramer (pH 7·0). Based on this fluorescence data and the already known crystal structure data, it appears that tryptophanyl 88 in concanavalin-A may be selectively excited by 305 nm radiation     

Immunological studies on erythrocyte phosphoglucomutase isozymes

Human erythrocytes (phenotype PGM1 a1 or PGM1 a3) contain two sets of phosphoglucomutase isozymes, produced by the expression of the PGM1 and and PGM2 loci. The two sets are constituted each by two forms, of which that called "secondary" is thought to derive from the post-translational modification of that called "primary". Cross-reactivities of these isozymes were studied by means of monospecific rabbit antibodies against purified human red cell PGM1 and PGM2 "primary" isozymes. The results show that the PGM1 and PGM2 forms are not immunologically related and provide further proof of the post-synthetic origin of "secondary" isozymes and of the multifunctionality of PGM2 phosphoglucomutases.     

Hydrogen-ion titration studies on erythrocyte membranes.

1. H+ titration was used to detect the presence of ionizable groups on human erythrocyte plasma membranes. Between pH2.9 and 11.3, two significant peaks of H+ association/dissociation occur in the differential from of the titration curve, one at pH3. 1. And the other at pH10.3. 2. After disruption of membrane structure by exposure to high pH or by the addition of sodium dodecyl sulphate, maxima of H+ association/dissociation were seen at pH3.1,4.3,6.5,10.3 and 10.7. 3. Spectrophotometric assay and selective chemical treatments were used to identify several of the titratable residues. 4. The degree of eleectrostatic interaction between titratable charged groups was investigated by comparing the titration characteristics of the membranes before and after modification of membrane structure.     

Further studies on sheep polymorphic erythrocyte diaphorase

Starch gel electrophoresis of sheep hemolysates revealed anodically faster, poly. morphic NADH/NADPH diaphorase (Dial) and slower NADH diaphorase (Dia2). Frequencies of alleles Dia1 F and Dia1 S for six sheep breeds in Czechoslovakia are given and efficacy for parentage control is discussed. A heterogeneity in Dia2 is caused by a prolonged storage of samples.     

Fluorescence polarization studies on myosin-substrate interaction

The degree of polarization of the intrinsic fluorescence of purified myosin was estimated. On addition of ATP, polarization of the fluorescence of myosin increased when excited at wavelengths longer than 300 nm. In kinetic studies, coupled with the decay of the increased intensity of fluorescence of myosin, the increased polarization of the fluorescence decreased when the ATP was depleted. The decay of the increased polarization of fluorescence of myosin was specific to MgATP. According to the theory of polarization of the fluorescence of proteins, it is likely that some tryptophan residues of myosin, which are responsible for the increase in the fluorescence intensity and polarization when myosin interacts with substrates, reduce their local freedom of rotation.     

Serum proteins and erythrocyte enzymes of populations in Iran

The genetic polymorphisms of nine biochemical genetic markers were investigated in four populations (Turks, Kurds, Tehranis and Kermanis) living in the north-east and south-east regions of Iran. Only one of the nine loci studied (acid phosphatase) showed significant gene frequency differences and for the C3 system the F allele frequencies in these populations were the lowest ever reported from Iran.