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Biomedical subjects

H Fasold

Publications and source records attributed to H Fasold.

At least 55 records · Page 3Linked to original sources
PubMed1982

Chemical modification of membrane proteins by brominated taurodehydrocholate in isolated hepatocytes; relationship to the uptake of cholate and of phalloidin and to the sensitivity of hepatocytes to phalloidin.

In vitro treatment of isolated rat hepatocytes with brominated taurodehydrocholic acid (BTC) reduced their sensitivity against phalloidin and inhibited the uptake of phalloidin as well as of cholate in an irreversible and concentration dependent manner. BTC was taken up itself by liver cells; this process was inhibited by 4,4'-diisothiocyano 2,2'-stilbene disulfonate (DIDS). When hepatocytes were incubated with 35S-BTC their plasma membranes contained five labeled protein species with molecular weights of 67,000, 49,000, 38,000, 32,000 and 24,000 as shown by SDS-electrophoresis. No marked difference was observed when isolated plasma membranes from livers were directly treated with the affinity label. DIDS suppressed covalent binding of 35S-BTC to membrane components drastically. Incubation of phalloidin insensitive AS-30D ascites hepatoma cells with 35S-BTC did not result in a chemical modification of the above five proteins. This agrees with an earlier observation that hepatoma cells are unable to take up phalloidin and bile acids (Petzinger et al. 1979; Rufeger and Grundmann 1977; Kroker et al. 1978).

Affinity Labels↗
PubMed1982

The kinetics of intramolecular cross-linking of the band 3 protein in the red blood cell membrane by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid (H2DIDS).

The two isothiocyanate groups of the anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2-2'-disulfonate (H2DIDS) may react covalently with two lysine residues called a and b that reside on the chymotryptic 60,000 Dalton and 35,000 Dalton segments, respectively, of the band 3 protein of the human erythrocyte membrane. Under suitable conditions, the reaction leads to the establishment of intramolecular cross-links between a and b (M.L. Jennings & H. Passow, 1979, Biochim. Biophys. Acta 554:498-519). In the present work, the time course of the reactions with a and b, and of the establishment of the cross-link were investigated experimentally and compared with simple mathematical models of the reaction sequence. The rates of reaction with a and b were found to increase with increasing pH. Regardless of pH, the rate of reaction with a exceeds that with b several-fold. Once the H2DIDS molecule has reacted with a, the rate of the subsequent reaction of the other isothiocyanate group with b is reduced by about 1/30. The reactions that follow the unilateral attachment to site b are not yet clear. A more detailed analysis of the time course of the cross-linking reaction suggests that a satisfactory description of the kinetics requires the assumption that the H2DIDS binding site may exist in two different states, and that the transition from one state to the other is associated with changes of the reactivities of either lys a alone or of both lys a and b. This led to the formulation of the two-states model of the H2DIDS binding site, which is supported by other pieces of independent evidence. The analysis of the pH dependence of the rate of thiocyanylation of b shows that the apparent pK value of that lysine residue is about 9.9 to 10.0 and hence slightly lower than the intrinsic pK of a lysine residue in an aqueous environment.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Characterization of an ATPase on the inside of rat-liver nuclear envelopes by affinity labeling.

Nuclear envelope membranes from rat liver cells contain ATPases, one of which can be inhibited and irreversibly labeled by (S-dinitrophenyl)-6-mercaptopurine riboside triphosphate. Inhibition and covalent substitution of the ATPase are achieved only after disruption of the nuclei, the ATP analogue is inactive on the ATPase activity of whole nuclei or on vesicles of the membrane prepared after a modified heparin method of Bornens and Courvalin. Electron micrographs and scanning micrographs helped to establish the characterization of closed vesicles and intact nuclei. With the aid of (alpha-32P)-labeled, and of the (beta, gamma-32P)-labeled analogue, it was possible to demonstrate the incorporation of the nucleotide into a few protein regions of the nuclear membrane disc electrophoresis pattern.

Adenosine Triphosphatases↗
PubMed1980

Anion transport across the red blood cell membrane and the conformation of the protein in Band 3.

Measuring the rate of dinitrophenylation of a specific lysine residue (called a) that is allosterically linked to the transfer site, it could be demonstrated that the anion transport protein may exist in two different conformational states, designated cis and trans. In the cis conformation a is easily accessible for reaction with dinitrofluorobenzene; in the trans conformation, a is less accessible. In the presence of the substrate anion Cl, the equilibrium between the cis and trans conformation is towards the cis conformation. Reversibly acting inhibitors of anion transport arrest the transport system, either predominantly in the cis or in the trans conformation. Phlorizin and certain positively charged derivatives of furosemide produce arrest in cis conformation, internal 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonate (APMB) and Ca++ in trans conformation. Within this frame of reference, the different susceptibilities of the transfer site to internal and external 4,4' diacetamido-2,2'-stilbene disulfonate (DAS) are interpreted on the assumption that the conformation of the transfer site changes during the transition of the transport protein from the cis to the trans conformation, so that in the trans conformation a reaction with DAS is no longer possible.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1980

Enhancement of anion equilibrium exchange by dansylation of the red blood cell membrane.

Dansylation of resealed red cell ghosts enhances the band 3 protein-mediated equilibrium exchange of sulfate ions. After dansylation, the specific anion transport inhibitor 4,4'-diisothiocyanato-dihydrostilbene 2,2'-disulfonate (H2DIDS) is still capable of combining with its original binding site on the band 3 protein and of producing the same high degree of inhibition of sulfate exchange as in the untreated red cell ghost. Nevertheless, dansylation causes allosteric effects at the H2DIDS-binding site that exhibit themselves by an increased susceptibility to dinitrophenylation of one of the amino acid residues that is involved in the covalent bond formation with H2DIDS and a decrease of the apparent KI values for two reversibly acting inhibitors that are known to produce their effects at the H2DIDS-binding site of the band 3 protein. The degree of enhancement of divalent anion exchange depends on both the pH that existed during dansylation and the pH at which the anion equilibrium exchange across the dansylated membrane is measured. The effect of dansylation reaches a broad maximum around ph 6.6. In untreated ghosts, divalent anion equilibrium exchange passes through a maximum around pH 6.3. After dansylation under optimal conditions at pH 6.6, anion equilibrium exchange as measured below the maximum of pH 6.3 is much less enhanced than above the maximum. Under suitable experimental conditions, the maximum may be replaced by a plateau that extends up to at least pH 8.5. At this pH, the enhancement is about 100-fold. Thus, the pH dependence of divalent anion exchange becomes more similar to that of monovalent anion exchange. The apparent activation enthalpies for sulfate-equilibrium exchange across the modified membrane, as measured at pH 6.3 and 7.9, are indistinguishable, independent of temperature between 0 and 37 degrees C and amount to 146 kj/mol. This is similar to the activation enthalpies measured in the untreated membrane. The mode of action of dansyl chloride is discussed on the basis of currently considered mechanisms of divalent anion transport, for which the pertinent equations are presented.

Anions↗
PubMed1978

The synthesis of a photoreactive puromycin analogue and its application for labeling proteins in the 50-S subunit of Escherichia coli ribosomes.

A photoreactive puromycin analogue, 6-dimethylamino-9-[3-(p-azido-L-beta-phenylalanylamino)-3-deoxy-beta-ribofuranosyl] purine, was synthesized. Biological activity was demonstrated by inhibition of the poly (U)-directed phenylalanine-incorporation system and by decomposition of isolated polysomes from Escherichia coli. The 3H-labeled puromycin analogue was covalently attached to the 50-S subunit of isolated 70-S ribosomes from Escherichia coli after irradiation. More than 90% of the radioactivity was bound to the protein fraction. The 70-S proteins were separated by two-dimensional gel electrophoresis. The proteins labeled primarily were those of the 50-S subunit, identified as L6, L13, L18, L22 and L25. On the basis of the affinity label used and supportive data from the literature, it is concluded that these proteins are at the active center of the 50-S particle and probably belong to the region of the ribosomal A site.

Affinity Labels↗
PubMed1978

Inhibitory effects of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) in the response of isolated hepatocytes to phalloidin.

4,4'-Diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) inhibits the typical development of protrusions, regularly seen after treatment of isolated hepatocytes with phalloidin. The degree of inhibition depends on the time of preincubation and on the concentration of DIDS, but not on the concentration of phalloidin. DIDS is more effective than H2DIDS. The inhibition by both compounds is irreversible. The binding capacity of hepatocytes for H2DIDS is much higher than that of the phalloidin-insensitive hepatoma cells. Gel electrophoresis of lysates from cells, pretreated with 3H2DIDS demonstrates that actin binds very little of the inhibitor. Our results suggest that a protein structure on the surface of hepatocytes, needed for the response to phalloidin, is influenced by DIDS or H2DIDS.

Animals↗
PubMed1977

The polymerization reaction of muscle actin.

Recent advances in the studies of the aggregation of G-actin monomers, containing one molecule of ATP, to long filaments of F-actin, with a concomitant hydrolysis of the nucleotide to ADP, are reviewed. With the aid of omega-ATP, the association and dissociation rate constant of the nucleotide could be determined. The binding of the nucleotide is enhanced by the binding of one Ca++ ion, probably at a different site. The delta G value of the Mg++ or Ca++ induced polymerization has been determined to --39 to--59 kJ/mole, the critical protein concentration for the ATP-G-actin to ADP-F-actin conversion is very strongly influenced by the concentration of bivalent cations. The rate constants of the protein monomers, and the rate and equilibrium constants for the propagation step show the process to be extremely cooperative. Actin shows the interesting phenomenon of translocational head-to-tail polymerization, which may be regulated by ATP. The contact sites between the monomers in F-actin have been labeled by chemical modification. Two tryosine residues, 53 and 69, are probably close to one of the two sites. The ATP binding sites has been labeled by an ATP analog, and there is evidence that it is close to the contact site.

Actins↗
PubMed1977

Comparison of hydrophobic and strongly hydrophilic cleavable crosslinking reagents in intermolecular bond formation in aggregates of proteins or protein-RNA.

Most of the bifunctional reagents in protein chemistry possess a strongly hydrophobic backbone, derived from aliphatic or aromatic hydrocarbons. Even bifunctionals of more than 30 A in length of this sort form intramolecular bridges preferentially. In recent years, the intermolecular crosslinking of physiological protein aggregates has gained in importance. As shown in the crosslinking of hemoglobin with two sets of hydrophobic and strongly hydrophilic reagents, derived from azo dyes and tartaric acid, respectively, in this case it is not primarily the length of the bifunctional, but the hydrophilic structure that will enhance intermolecular crosslinking. Artificial dimers of native structure may be obtained. For the crosslinking of RNA to protein, we have synthesized a new reagent, 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid p-nitrophenyl ester. In a two step reaction, it is attached to adenine and cytosine moieties at pH 6 first, and to lysine side chains at pH 7,5. The reagent has been applied to the poly-A sequence of globin messenger RNA nucleoprotein.

Adenosine↗
PubMed1976

A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS).

DIDS (4,4'-diisothiocyano stilbene-2,2'-disulfonic acid) and H2DIDS (4,4'-diisothiocyano-1,2-diphenyl ethane-2,2'-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974. J. Cell Biol. 62:1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and greater 60% of DIDS binding were rapid, independent of temperature. Slow H2-DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1-1.2, concentration-dependent. Under the conditions studied (0-50 muM, hematocrit 10%, 5-37 degrees C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS. Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases, H2DIDS inhibition was complete at 1.9 X 10(6) or 1.2 X 10(6) molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3 X 10(6) and 1.1 X 10(6). It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.

Benzenesulfonates↗