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Biomedical subjects

V Bennett

Publications and source records attributed to V Bennett.

At least 127 records · Page 7Linked to original sources
PubMed1985

Plasmodium falciparum malaria: band 3 as a possible receptor during invasion of human erythrocytes.

Human erythrocyte band 3, a major membrane-spanning protein, was purified and incorporated into liposomes. These liposomes, at nanomolar concentrations of protein, inhibited invasion of human erythrocytes in vitro by the malaria parasite Plasmodium falciparum. Liposomes containing human band 3 were ten times more effective in inhibiting invasion than those with pig band 3 and six times more effective than liposomes containing human erythrocyte glycophorin. Liposomes alone or liposomes containing erythrocyte glycolipids did not inhibit invasion. These results suggest that band 3 participates in the invasion process in a step involving a specific, high-affinity interaction between band 3 and some component of the parasite.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1985

Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes.

Brain membranes contain an actin-binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purified from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease-resistant domains of Mr = 72,000 that contain spectrin-binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrum beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin-associated actin filaments. Another spectrin-binding protein has been purified from brain membranes, and this protein cross-reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP-dependent and Ca/calmodulin-dependent protein kinases with equivalent physical properties, immunological cross-reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.

Animals↗
PubMed1985

Human erythrocyte myosin: identification and purification.

Human erythrocytes contain an Mr 200,000 polypeptide that cross-reacts specifically with affinity-purified antibodies to the Mr 200,000 heavy chain of human platelet myosin. Immunofluorescence staining of formaldehyde-fixed erythrocytes demonstrated that the immunoreactive myosin polypeptide is present in all cells and is localized in a punctate pattern throughout the cell. Between 20-40% of the immunoreactive myosin polypeptide remained associated with the membranes after hemolysis and preparation of ghosts, suggesting that it may be bound to the membrane cytoskeleton as well as being present in the cytosol. The immunoreactive myosin polypeptide was purified from the hemolysate to approximately 85% purity by DEAE-cellulose chromatography followed by gel filtration on Sephacryl S-400. The purified protein is an authentic vertebrate myosin with two globular heads at the end of a rod-like tail approximately 150-nm long, as visualized by rotary shadowing of individual molecules, and with two light chains (Mr 25,000 and 19,500) in association with the Mr 200,000 heavy chain. Peptide maps of the Mr 200,000 heavy chains of erythrocyte and platelet myosin were seen to be nearly identical, but the proteins are distinct since the platelet myosin light chains migrate differently on SDS gels (Mr 20,000 and 17,000). The erythrocyte myosin formed bipolar filaments 0.3-0.4-micron long at physiological salt concentrations and exhibited a characteristic pattern of myosin ATPase activities with EDTA, Ca++, and Mg++-ATPase activities in 0.5 M KCl of 0.38, 0.48, and less than 0.01 mumol/min per mg. The Mg++-ATPase activity of erythrocyte myosin in 0.06 M KCl (less than 0.01 mumol/min per mg) was not stimulated by the addition of rabbit muscle F-actin. The erythrocyte myosin was present in about 6,000 copies per cell, in a ratio of 80 actin monomers for every myosin molecule, which is an amount comparable to actin/myosin ratios in other nonmuscle cells. The erythrocyte myosin could function together with tropomyosin on the erythrocyte membrane (Fowler, V.M., and V. Bennett, 1984, J. Biol. Chem., 259:5978-5989) in an actomyosin contractile apparatus responsible for ATP-dependent changes in erythrocyte shape.

Antibodies↗
PubMed1984

Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel.

Brain ankyrin was purified from pig brain membranes in milligram quantities by a procedure involving affinity chromatography on erythrocyte spectrinagarose. Brain ankyrin included two polypeptides of Mr = 210,000 and 220,000 that were nearly identical by peptide mapping and were monomers in solution. Brain ankyrin and erythrocyte ankyrin are closely related proteins with the following properties in common: 1) shared antigenic sites, 2) high-affinity binding to the spectrin beta subunit at the midregion of spectrin tetramers, 3) a binding site for the cytoplasmic domain of the erythrocyte anion channel, 4) a binding site for tubulin, 5) a similar domain structure with a protease-resistant domain of Mr = 72,000 that contains the spectrin-binding activity and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg of membrane protein in demyelinated membranes based on radioimmunoassay with antibody raised against brain ankyrin and affinity purified on brain ankyrin-agarose. Brain spectrin tetramers are present at 30 pmol/mg of membrane protein. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes. Brain ankyrin also may attach microtubules to membranes independently of spectrin and has the potential to interconnect microtubules and spectrin-associated actin filaments.

Animals↗
PubMed1984

Erythrocyte membrane tropomyosin. Purification and properties.

Two polypeptides of Mr approximately 29,000 and 27,000 have been identified in human erythrocyte membranes that cross-react specifically with affinity purified antibodies to chicken gizzard tropomyosin. The cross-reacting polypeptides are quantitatively retained on the membrane after cell lysis if millimolar concentrations of magnesium are included in the lysis and wash buffers, indicating that they are membrane-bound proteins under physiological conditions. Milligram quantities of these immunoreactive polypeptides have been purified to greater than 95% purity from a low salt extract of membranes by DEAE-chromatography, precipitation at pH 4.4, and heating to 85 degrees C to denature contaminants. Physical similarities of the erythrocyte protein to other tropomyosins include (a) amino acid composition (b) anomalous migration of the Mr approximately 29,000 and 27,000 polypeptides on sodium dodecyl sulfate-gels in the presence of 6 M urea to apparent Mr approximately 43,000 and 38,000, respectively (c) arrangement of chains as dimers of Mr approximately 60,000 based on cross-linking studies and calculation of molecular weight from hydrodynamic values (Rs = 5.9 nm, sedimentation coefficient = 2.5 S; partial specific volume = 0.72 cm3/g) and (d) highly asymmetric shape, based on a frictional ratio of 2.07. Binding of erythrocyte tropomyosin to muscle F-actin saturates at one tropomyosin molecule (Mr approximately 60,000) to 6-7 actin monomers and is highly cooperative with a Hill coefficient of about 2.8, similar to muscle tropomyosins. Binding also exhibits a high degree of cooperativity as a function of the magnesium concentration with a transition between no binding and complete binding between 1 and 2 mM MgCl2. Increasing the magnesium concentration from 2 to 10 mM increases the apparent affinity of tropomyosin for actin from approximately 2.6 X 10(6) M-1 to approximately 2.7 X 10(7) M-1 without effect on the Hill coefficient. The tropomyosin polypeptides comprise about 1% of the erythrocyte membrane protein and are present in a ratio of one Mr approximately 60,000 tropomyosin molecule to 7-8 actin monomers, an amount almost sufficient to coat all of the F-actin on the membrane. These data are consistent with the association of two tropomyosin molecules with each of the short actin filaments (12-17 monomers long) thought to exist in the erythrocyte membrane cytoskeleton. The erythrocyte tropomyosin could function to mechanically stabilize these actin filaments as well as play a role in regulating the interaction of spectrin with actin.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1984

Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain.

Polypeptides of Mr = 190,000-220,000 that cross-react with erythrocyte ankyrin were detected in immunoblots of membranes from pig lens, pig brain, and rat liver. The cross-reacting polypeptides from brain were cleaved by chymotrypsin to fragments of Mr = 95,000 and 72,000 which are the same size as fragments obtained with erythrocyte ankyrin. The brain 72,000 Mr fragment associated with erythrocyte spectrin, and the binding occurred at the same site as that of erythrocyte ankyrin 72,000 Mr fragment since (a) brain 72,000 Mr fragment was adsorbed to erythrocyte spectrin-agarose and (b) 125I-labeled erythrocyte spectrin bound to brain 72,000 Mr fragment following transfer of the fragment from a sodium dodecyl sulfate gel to nitrocellulose paper, and this binding was displaced by erythrocyte ankyrin 72,000 Mr fragment. Brain 72,000 Mr fragment was purified about 400-fold by selective extraction and by continuous chromatography on columns attached in series containing DEAE-cellulose followed by erythrocyte spectrin coupled to agarose, and finally hydroxylapatite. The brain 72,000 Mr fragment was not derived from contaminating erythrocytes since peptide maps of pig brain and pig erythrocyte 72,000 Mr fragments were distinct. The amount of brain 72,000 Mr fragment was estimated as 0.28% of membrane protein or 39 pmol/mg based on radioimmunoassay with 125I-labeled brain fragment and antibody against erythrocyte ankyrin. Brain spectrin tetramer was present in about the same number of copies (30 pmol/mg of membrane protein) based on densitometry of Coomassie blue-stained sodium dodecyl sulfate gels. The binding site on brain spectrin for both brain and erythrocyte ankyrin 72,000 Mr fragments was localized by electron microscopy to the midregion of spectrin tetramers about 90 nM from the near end and 110 nM from the far end. These studies demonstrate the presence in brain membranes of a protein closely related to erythrocyte ankyrin, and are consistent with a function of the brain ankyrin as a membrane attachment site for brain spectrin.

Animals↗
PubMed1984

Bepridil and cetiedil. Vasodilators which inhibit Ca2+-dependent calmodulin interactions with erythrocyte membranes.

Two new vascular smooth muscle relaxants, bepridil and cetiedil, were found to possess specific CaM-inhibitory properties which resembled those of trifluoperazine. Trifluoperazine, bepridil, and cetiedil inhibited Ca2+-dependent 125I-CaM binding to erythrocyte membranes and CaM activation of membrane Ca2+-ATPase with IC50 values of approximately 12, approximately 17, and approximately 40 microM, respectively. This does not appear to be the result of a nonspecific hydrophobic interaction since inhibition was not observed with micromolar concentrations of many other hydrophobic agents. The predominant inhibition of binding and Ca2+-ATPase activation was competitive with respect to CaM. Bepridil and cetiedil bind directly to CaM since these drugs displaced [3H]trifluoperazine from sites on CaM. Inhibition of Ca2+-ATPase and binding by the drugs was not due to interference with the catalytic activity of this enzyme since: (a) neither inhibition of CaM-independent basal Ca2+-ATPase activity nor inhibition of proteolytically-activated Ca2+-ATPase activities were produced by these agents, and (b) no drug-induced inhibition of CaM binding was detected when membranes were preincubated with these agents but washed prior to addition of 125I-CaM. Thus, bepridil and cetiedil competitively inhibit Ca2+-dependent interactions of CaM with erythrocyte membranes, most likely by a direct interaction between these drugs and CaM. The principal clinical actions of these drugs may be explained by their interactions with CaM or CaM-related proteins leading to reduced activation of Ca2+-regulated enzymes in certain other tissues, such as myosin light chain kinase in vascular smooth muscle.

Adult↗
PubMed1984

Proteins closely related to spectrin and ankyrin are general components of cell membranes.

Membrane-associated analogues of erythrocyte spectrin and ankyrin have been detected in nonerythroid cells by crossreaction with antibodies. Brain spectrin and a spectrin-binding domain of brain ankyrin have been purified and demonstrated to have all known functional activities of their erythrocyte counterparts. The nonerythroid forms of spectrin and ankyrin may have functions such as providing support for the lipid bilayer, mediating some types of actin-membrane interactions, and integrating membrane-spanning proteins with cytoplasmic structural proteins.

Actins↗
PubMed1983

Brain spectrin. Isolation of subunits and formation of hybrids with erythrocyte spectrin subunits.

Brain spectrin tetramer was purified from pig brain membranes in milligram quantities. The tetramer had subunits of Mr = 265,000 (alpha) and Mr = 260,000 (beta), Rs = 21.4 nM, S20,w = 11 S, V = 0.725 ml/g, frictional ratio of 2.9, and calculated molecular weight of 9.7 x 10(5). The subunits were isolated in greater than 95% purity by chromatography on hydroxylapatite in 7 M urea, as described for erythrocyte spectrin (Calvert, R., Bennett, P., Gratzer, W. (1980) Eur. J. Biochem. 107, 355-361). Peptide maps of the subunits revealed few if any common peptides. The subunits were visualized by rotary shadowing as single-stranded flexible rods 100 nm in length with no homodimers by lateral or end association. When the subunits were renatured together, double-stranded tetramers 200 nm in length were formed, as well as higher oligomers. These results indicate that spectrin tetramers are formed by laterally associated alpha,beta dimers attached by head-to-head linkage of each alpha chain with a beta chain. The reassembled subunits regained the ability to increase the low shear viscosity of actin, although isolated alpha or beta subunits were inactive. Hybrid molecules were formed with brain alpha and pig erythrocyte spectrin beta subunit and were visualized as double-stranded rods 100 nm long with no tetramers. 125I-labeled brain alpha chain that was hybridized with erythrocyte beta subunit acquired the ability to bind to ankyrin sites on erythrocyte membranes. 125I-labeled brain alpha chain bound only to beta subunits of erythrocyte and brain spectrin following transfer of these polypeptides to nitrocellulose paper from sodium dodecyl sulfate gels. Affinity-purified antibodies against brain spectrin cross-reacted with both subunits of erythrocyte spectrin and with polypeptides of a similar molecular weight to brain spectrin in membranes of all tissues examined. Cross-reacting polypeptides were localized exclusively in plasma membranes of subcellular fractions from liver. These studies provide strong additional evidence that brain and mammalian erythrocyte spectrin belong to a closely related family of proteins with conserved functions in both subunits.

Actins↗
PubMed1983

Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes.

This report describes Ca2+-dependent binding of 125I-labeled calmodulin (125I-CaM) to erythrocyte membranes and identification of two new CaM-binding proteins. Erythrocyte CaM labeled with 125I-Bolton Hunter reagent fully activated erythrocyte (Ca2+ + Mg2+)-ATPase. 125I-CaM bound to CaM depleted membranes in a Ca2+-dependent manner with a Ka of 6 x 10(-8) M Ca2+ and maximum binding at 4 x 10(-7) M Ca2+. Only the cytoplasmic surface of the membrane bound 125I-CaM. Binding was inhibited by unlabeled CaM and by trifluoperazine. Reduction of the free Ca2+ concentration or addition of trifluoperazine caused a slow reversal of binding. Nanomolar 125I-CaM required several hours to reach binding equilibrium, but the rate was much faster at higher concentrations. Scatchard plots of binding were curvilinear, and a class of high affinity sites was identified with a KD of 0.5 nM and estimated capacity of 400 sites per cell equivalent for inside-out vesicles (IOVs). The high affinity sites of IOVs most likely correspond to Ca2+ transporter since: (a) Ka of activation of (Ca2+ + Mg2+)-ATPase and KD for binding were nearly identical, and (b) partial digestion of IOVs with alpha-chymotrypsin produced activation of the (Ca2+ + Mg2+)-ATPase with loss of the high affinity sites. 125I-CaM bound in solution to a class of binding proteins (KD approximately 55 nM, 7.3 pmol per mg of ghost protein) which were extracted from ghosts by low ionic strength incubation. Soluble binding proteins were covalently cross-linked to 125I-CaM with Lomant's reagent, and 2 bands of 8,000 and 40,000 Mr (Mr of CaM subtracted) and spectrin dimer were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. The 8,000 and 40,000 Mr proteins represent a previously unrecognized class of CaM-binding sites which may mediate unexplained Ca2+-induced effects in the erythrocyte.

Ca(2+) Mg(2+)-ATPase↗
PubMed1983

Spectrin and ankyrin in brain.

Further similarity between mammalian erythrocyte spectrin and pig brain spectrin has been demonstrated by (a) formation of hybrid molecules with brain alpha-chains and erythrocyte beta-chains and by (b) identification of an ankyrin protein in brain membranes. Hybrid spectrin molecules prepared from brain alpha-chains and erythrocyte beta-chains were visualized by low-angle rotary shadowing as double-stranded rods (dimers) 100 nM in length. 125I-labeled brain alpha-chain that was hybridized with erythrocyte beta-subunit acquired ability to bind to ankyrin sites on erythrocyte membranes. 125I-labeled brain alpha-chain bound only to beta-subunits of erythrocyte and brain spectrin following transfer of these polypeptides to nitrocellulose paper from sodium dodecyl sulfate (SDS) gels. Thus brain spectrin and mammalian erythrocyte spectrin have shared functional sites involved in association of their subunits. Additional evidence for similarity of brain and erythrocyte membranes is the finding of a 210,000 Mr membrane protein in brain that cross-reacts with erythrocyte ankyrin and has a water-soluble domain of 72,000 Mr that is produced by protease digestion. The 72,000 Mr domain of brain ankyrin has been isolated by affinity chromatography on erythrocyte spectrin-Sepharose, and was demonstrated to bind directly to erythrocyte and brain spectrin. The brain 72,000 Mr fragment has distinct peptide maps from the erythrocyte 72,000 Mr ankyrin fragment and thus is not a result of erythrocyte contamination.

Animals↗
PubMed1983

Detection of pulmonary venous flow by pulsed Doppler echocardiography in children.

Parasternal pulsed Doppler echocardiographic examinations of the left atrium were prospectively performed in 14 pediatric patients, aged 2 weeks to 8 years, in order to characterize the left atrial flow pattern in children. None of the patients had clinical or cardiac catheterization evidence (9 of 14 patients) of either mitral regurgitation, right-to-left shunts at the atrial level, or anomalies of pulmonary venous drainage. In all 14 patients, a systolic-diastolic positive velocity pattern could be recorded within the left atrium. Furthermore, this pattern was more readily detected in those children with left-to-right shunts. We conclude that continuous positive velocity patterns are commonly recorded in the left atria of children and probably represent pulmonary venous return. Demonstration of this signal is contingent on proper orientation of the transducer with respect to pulmonary venous flow.

Child↗
PubMed1982

Brain spectrin, a membrane-associated protein related in structure and function to erythrocyte spectrin.

An immunoreactive analogue of erythrocyte spectrin has been purified from brain membranes. This protein co-sediments with and cross-links actin filaments, associates with spectrin-binding sites on erythrocyte membranes, and has been visualized by rotary shadowing as an extended, flexible rod. The brain spectrin comprises 3% of the total membrane protein, and may have a major role in mediating linkage of actin to membranes.

Actins↗