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Biomedical subjects

M Shinitzky

Publications and source records attributed to M Shinitzky.

At least 109 records · Page 6Linked to original sources
PubMed1978

Peturbation of lymphocyte response to concanavalin A by exogenous cholesterol and lecithin.

Incubation of lymphocytes with lecithin liposomes enriched with cholesterol, elevated the cholesterol level of the cells relative to phospholipids. Treatment of lymphocytes with pure lecithin liposomes resulted in the converse effect. Both these treatments resulted in suppression of the induction phase of the response to concanavalin A and were practically reversible. It is suggested that these changes induce modulations of the fluidity of the lymphocyte membrane which may also take place in vivo by serum lipoproteins. Based on this study, the possible effects of lipids on lymphocyte activation are discussed.

Animals↗
PubMed1977

The effect of phosphatidylcholine to sphingomyelin mole ratio on the dynamic properties of sheep erythrocyte membrane.

Sheep red blood cells are shown to incorporate phosphatidylchline when incubated in human plasma in the presence of EGTA. This treatment results in up to a 5-fold increase in mol ratio of phosphatidylcholine to sphingomyelin. By replacing EGTA with Ca+ the increase of phsphatidylcholine content is completely inhibited, due to the activation of the membrane bound lecithinase which rapidly degrades the incorporated phosphatidylcholine. Analogous treatments of the isolate membranes resulted in similar phosphatidylcholine incorporation but in the presence of Ca+ a residual phosphatidylcholine uptake was still oberved. These results suggest that in the isolated membranes small amounts of phosphatidylcholine can be incorporated into an additional region which is unavailable for the membrane lecithinase. The increase in the phosphatidylcholine to sphingomyelin mol ratio in sheep red blood cells is concomitant with an increase in lipid fluidity, as well as increase in osmotic fragility9

Animals↗
PubMed1977

Degree of exposure of membrane proteins determined by fluorescence quenching.

Quaternary salts of 4-picoline are shown to act as efficient quenchers of tryptophan fluorescence in membrane proteins. Fluorescence quenching determinations of sarcoplasmic reticulum membranes from rabbit muscle and of human erythrocyte membranes of different cholesterol to phospholipid mole ratios (C/PL) were carried out with quaternary picolinium salts in phosphate-buffered saline (PBS) and in 2,2,2-trifluorethanol (TFE)-water 2:1 (v/v), where the membrane is presumably completely disintegrated. In both solvent systems, the tryptophan quenching characteristics were typical of heterogenous systems and were analyzed as such. The ratio of the fraction of fluorescence intensity available for quenching with N-methylpicolinium perchlorate in PBS and in 2:1 TFE-water, (formula: see text), was taken as an index for the bulk degree of exposure of the membrane proteins to the aqueous surrounding. This value was found to increase with C/PL which is in line with the notion that increase in lipid microviscosity results in increase of exposure of membrane proteins. Analogous experiments were performed with N-hexyl- and N-benzylpicolinium, which can quench tryptophyl residues in both the aqueous phase and the hydrocarbon-water interface, and with N-hexadecylpicolinium which is dissolved in the membrane lipid layer and acts mostly as a static quencher of tryptophan at the hydrocarbon-water interface. With these quenchers the complementary indices (formula: see text) and (formula: see text), which represent the fraction of the protein mass located in the hydrocarbon-water interface and in the hydrocarbon layer, respectively, could be semiquantitatively resolved.

Animals↗
PubMed1977

Microviscosity modulation during the cell cycle of neuroblastoma cells.

Microviscosity (n) of the cell membrane lipid layer was determined in synchronized C1300 mouse neuroblastoma cells (clone Neuro-2A) by fluorescence polarization of 1,6-diphenystratum. The determined n value was maximal in mitosis, decreased markedly in the G1 phase, remained constant at a low level during the S phase, and increased again during the G2 phase. These findings imply a direct role of the cell membrane fluidity in regulation of the cell cycle.

Animals↗
PubMed1977

Fluorescence polarization studies of rat intestinal microvillus membranes.

Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin.

Animals↗
PubMed1976

Structure-activity relationships of luliberin substituted at position 8.

Substitution of arginine at position 8 of luliberin by the basic amino acids homoarginine, lysine and diaminobutyric acid resulted in analogues in which the luteinizing hormone-releasing activity is markedly reduced, whereas the cross reactivity with specific antibodies to luliberin is preserved. Fluorimetric titrations of these analogues, carried out as with luliberin, revealed pK values of 6.00 +/- 0.05 and of 9.75 +/- 0.15 for His 2 and Try 5 respectively which are essentially the same as in luliberin. However, the rate of collisions between the side chains of His 2 and Trp 3 in these analogues was found to decrease by 36-39%. Substitution at position 8 with the non-basic amino acid omega-nitro arginine yielded an analogue possessing a very low hormonal activity as well as poor recognition of antibodies specific to luliberin. The fluorescence properties of this peptide are markedly different from those of luliberin and its three basic analogues. These results indicate that the functional integrity of the active unit His 2 . . . Tyr 5 . . . Arg 8 in luliberin depends both on size and basicity of the amino acid side chain at position 8.

Amino Acids, Diamino↗
PubMed1976

The interaction between retinol-binding proteins and prealbumins studied by fluorescence polarization.

The interaction between retinol-binding proteins and prealbumins of human and chicken was studied by fluorescence polarization techniques. The binding affinity between chicken plasma retinol-binding protein and chicken prealbumin was essentially the same as between the respective human proteins. Human urine retinol-binding protein displayed a similar affinity, though possibly slightly smaller than that of the human plasma protein, toward human prealbumin. Retinol-binding proteins and prealbumins of human and chicken have been found to cross-interact displaying an affinity similar to that displayed by the proteins of the same species. Solution of a binding equation which assumes identical, independent sites, indicated that the number of binding sites on prealbumin for retinol-binding protein is somewhat less than 2 with the human system, and in the neighborhood of 4 with the chicken system. A possible interpretation suggests that prealbumin possesses four identical binding sites for retinol-binding protein, one for each subunit, but that the binding is of a negative cooperative nature. A major share of the negative cooperativity is likely to result from steric hindrance induced by already bound retinol-binding protein molecules, which have a sizable volume compared to the volume of the prealbumin molecule. The cooperativity is likely to be more pronounced with the human system. Rotational relaxation times derived from Perrin plots suggest that 1:1 molecular complexes of retinol-binding proteins with prealbumins have a compact structure.

Animals↗
PubMed1976

Structural features of luliberin (luteinising hormone-releasing factor) inferred from fluorescence measurements.

The fluorescence and excitation spectra of luliberin (luteinizing hormone-releasing factor) in 0.005 M aqueous ammonium acetate are identical in shape to those of N-acetyltryptophan amide and are related to the indole side chain of Trp3. The change of fluoresecence intensity of luliberin with pH was measured in the range of pH 4-11. The increase of pH from 4 to 7.5 is followed by about 50% increase in fluorescence intensity due to deprotonation of the imidazolium side chain of His2. The fluorimetric titration curve in this pH region reveals a pK value for His2 of 5.95. Increasing of pH from 8 to 11 results in about 40% quenching of the fluorescence due to electronic energy transfer from the excited indole of Trp3 to the phenolate side chain of Tyr5. The pK value of Tyr5, obtained independently from the fluorimetric and photometric titrations indicate that at pH 7-8 luliberin contains only one charged residue, Arg8, which is in close vicinity to both His2 and Tyr5. The side chains of His2, Tyr5 and Arg8 presumably form a combined unit which may play an active role in the hormone action. Trp3 is at a maximal distance from this unit and may thus act as an independent active unit.

Gonadotropin-Releasing Hormone↗
PubMed1976

Binding affinities of retinol and related compounds to retinol binding proteins.

Fluorimetric titrations were used to determine apparent dissociation constants of the all-trans isomers of retinol, retinoic acid, retinyl acetate and retinyl palmitate to human-retinol binding protein and chicken-retinol binding protein. Enhancement of the fluorescence of retinol and retinyl acetate when bound to the protein was utilized to establish the binding affinity of these compounds. With retinoic acid which is essentially a non-fluorescent compound, quenching of protein fluorescence due to energy transfer to the bound ligand from tryptophanyl residues served to determine the binding affinity. The various ligands display 1:1 molecular complexes with both types of retinol binding proteins. Retinol, retinoic acid and retinyl acetate were found to have similar binding affinities to both species of carrier proteins: For retinol K'd=1.9 X 10(-7) M with human-retinol binding protein and K'd=1.5 X 10(-7) M with chicken-retinol binding protein; for retinoic acid K'd-2.1 X 10(-7) M with human-retinol binding protein; for retinyl acetate and K'd=2.2 X 10(-7) M with chicken-retinol binding protein; for retinyl acetate K'd=2.2 X 10(-7) M with human-retinol binding protein and K'd=1.7 X 10(-7) M with chicken-retinol binding protein. Retinyl palmitate appeared to have weak association with either of the two retinol binding proteins, if at all. The above results suggest that both human and chicken retinol binding proteins behave similar with respect to the binding of the ligands. Non-polar interactions probably play a primary role in the binding and effects of functional groups and charges are of secondary importance.

Animals↗
PubMed1976

Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.

Animals↗
PubMed1976

Vertical displacement of membrane proteins mediated by changes in microviscosity.

Membrane proteins of intact human erythrocytes were labeled with two fluorescent sulfhydryl reagents. The tagged cells were then subjected to simultaneous liposome treatments for either depletion or enrichment of membrane cholesterol content. Cholesterol depletion, which reduces membrane microviscosity, was followed by a series of fluorescence changes all indicating masking of the membrane proteins. Conversely increasing the membrane microviscosity by cholesterol enrichment resulted in an appreciable increase of the protein exposure to the aqueous surrounding. These findings strongly suggest that membrane proteins may be vertically displaced upon changes in lipid fluidity, a mechanism that may play a significant role in modulation of antigens and receptors in vivo.

Cholesterol↗
PubMed1976

A new method for assessment of fetal lung maturity.

A new method for evaluation of fetal lung maturity in utero is described. The method is based on the fluorescence polarization (P) of the lipids in the amniotic fluid after labelling with a special dye. Tests performed with 49 amniotic fluid samples drawn from 33 pregnancies clearly demonstrated a decrease in P during gestation which correlated with the increase in the lecithin/sphingomyelin ratio (L/S) in the fluid. During gestation the P value at 24 degrees C decreased steadily from about 0-4 to 0-2 and the value of P (24 degrees C) = 0-310 has been tentatively chosen as the threshold above which respiratory distress syndrome may develop. The described method, which requires a specially designed instrument, offers a series of important advantages over the presently available methods. It is simple, rapid, highly accurate and reproducible, and independent of amniotic fluid volume. The P value reflects the microviscosity in the whole lipid assembly of amniotic fluid and is not confined specifically to lecithin.

Amniotic Fluid↗