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Biomedical subjects

A Rothstein

Publications and source records attributed to A Rothstein.

At least 127 records · Page 7Linked to original sources
PubMed1975

Pyridoxal phosphate. An anionic probe for protein amino groups exposed on the outer and inner surfaces of intact human red blood cells.

Pyridoxal phosphate is a potent probe for exploring the "sidedness" of proteins in the membrane of the intact red blood cell. It reacts with amino groups with a high degree of specificity, forming a Schiff's base that can be fixed as an irreversible bond upon reduction with NaBH4; its binding site can be identified by use of [3-H]pyridoxal phosphate or NaB3-H4; it can be used as a surface probe under conditions of minimal penetration, or it can be used as a probe for proteins on the inside of the membrane under conditions of substantial uptake. Pyridoxal phosphate uptake involves a rapid and a slow component. The former represents the binding to the outer surface of the membrane; it is not substantially affected by pH and temperature, but is reduced considerably by pretreatment of cells by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid, a specific inhibitor of anion transport. The slow component represents penetration into the cell; it is blocked by high pH, low temperature, or pretreatment with the disulfonic stilbene. Pyridoxal phosphate itself is also an effective and specific inhibitor of the permeation of other anions. Under conditions of minimal uptake, the only labeled proteins are three glycoproteins and a protein of apparent molecular weight 95,000. Under conditions of substantial uptake into the cell, the other major protein bands seen by staining on acrylamide gels after electrophoresis are labeled. It is concluded that virtually all of the major membrane proteins interact with pyridoxal phosphate from one side of the membrane or the other. The differences in labeling under conditions of minimal or maximal uptake can, therefore, be attributed to the sidedness in the distribution of the membrane proteins rather than to differences in their reactivity.

Amino Acids↗
PubMed1975

A comparison of intact human red blood cells and resealed and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes.

Resealed ghosts and intact red blood cells were directly compared with respect to their interactions with surface proteins by 4.4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and by pyridoxal phosphate-borohydride (as seen after sodium dodecylsulfate/acrylamide gel electrophoresis) was substantially the same in cells and resealed ghosts under conditions in which a relatively small change would be apparent. In each membrane system, DIDS labels a protein component of apparent molecular weight 95 000 and pyridoxal phosphate labels the same protein plus three glucoprotein components. The sensitivity of surface proteins and of DIDS and pyridoxal phosphate-labelled sites to pronase was also similar in the cells and resealed ghosts. The glycoproteins were digested, in each case, and the 95 000 (molecular weight) protein was largely split into two proteins of apparent molecular weights 65 000 and 35 000, with both portions containing DIDS and pyridoxal phosphate in the presence of hemoglobin was similar to the labelling of intact cells, provided that the pyridoxal phosphate was present on both the outside and inside of the cells. Virtually all of the major protein components visible by staining on acrylamide gels were labelled. It is concluded that none of the probes could detect any substantial differences in reactivity of proteins of the outer surface of the membrane protein conformation or arrangement occur as a consequence of lysis and resealing of ghosts, that are detectable by the reported procedures.

Blood Proteins↗
PubMed1974

Relationship of cation influxes and effluxes in yeast.

The Na(+) efflux from Na(+)-rich yeast cells into a cation-free medium is largely balanced by the excretion of organic anions. In the presence of Rb(+), K(+), or high levels of H(+) (pH 3-4), the Na(+) efflux is increased and the organic anion excretion is suppressed so that stoichiometric cation exchanges occur. H(+) participates in the exchanges, moving into or out of the cells depending on the external pH and on the concentration of external Rb(+)(K(+)). The total cation efflux is dependent on the external Rb(+) concentration in a "saturation" relationship, but the individual cations in the efflux stream are not. The discrimination factor in the efflux pathway between H(+) and Na(+) is very large (of the order of 10,000), and between Na(+) and K(+) considerable (of the order of 50). For the latter pair, the recycling of K(+) from the cell wall space is an important factor in the discrimination. In addition, the Na(+) efflux as a function of Na(+) content follows a sigmoidal curve so that the discrimination factor is increased at high levels of cellular Na(+). Although the influx and efflux pathways behave as a tightly coupled system, the mechanism of coupling is not entirely clear. A single system with different cation specificities and kinetic behaviors on the inside and outside faces of the membrane could account for the data.

Biological Transport↗
PubMed1971

Chemical modification of membranes. I. Effects of sulfhydryl and amino reactive reagents on anion and cation permeability of the human red blood cell.

Four different amino-reactive reagents, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS),(1) 1-fluoro-2,4-dinitrobenzene (FDNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and 2-methoxy-5-nitrotropone (MNT) decrease the anion permeability of the human red blood cell, as measured by sulfate fluxes, whereas the sulfhydryl agent, parachloromercuriphenyl sulfonic acid (PCMBS), does not. In contrast, PCMBS increases the cation permeability as measured by K(+) leakage, whereas SITS does not. Of the other agents, FDNB increases the cation permeability to the same extent as PCMBS but MNT and TNBS produce smaller increases. PCMBS does not protect against FDNB as it does against other sulfhydryl agents (X-irradiation) and the FDNB effect on cations is attributed to amino groups. Studies of the binding of SITS indicate that it does not penetrate into the membrane and its failure to influence cation permeability is attributed to its inability to reach an internal population of amino groups. It is concluded that two ion permeability barriers, both involving proteins, are present in the red blood cell. The more superficial barrier contains amino groups and controls anion flow; the more internal barrier contains sulfhydryl and amino groups and controls cation flow. The amino groups contribute to the control of permeability by virtue of their positive charges, but the role of sulfhydryl groups is not clear. Only a small fraction of the membrane protein amino and sulfhydryl is involved in the barriers.

Antimony↗
PubMed1971

Chemical modification of membranes. II. Permeation paths for sulfhydryl agents.

The amino-reactive reagent, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS),(1) considerably reduces the uptake of the sulfhydryl agent, parachloromercuriphenylsulfonic acid (PCMBS), but does not reduce its effects on cation permeability and on cation transport. These data indicate that PCMBS enters the membrane by at least two channels, one sensitive and the other insensitive to SITS, with only the latter leading to the cation-controlling sulfhydryl groups. Substitution of phosphate or sulfate for chloride results in an inhibition of PCMBS uptake via the SITS-insensitive pathway. These and other data lead to the conclusion that the SITS-sensitive pathway is the predominant one for anion permeation, and the insensitive one for cation permeation. Parachloromercuribenzoate (PCMB), an agent that is more lipid-soluble than PCMBS, penetrates faster but has a smaller effect on cation permeability. Its uptake is less sensitive to SITS. These and other observations suggest that the cation permeation path involves an aqueous channel in the membrane.

Cell Membrane Permeability↗