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Biomedical subjects

A G Booth

Publications and source records attributed to A G Booth.

At least 19 recordsLinked to original sources
PubMed2001

Visualizing protein conformational changes on a personal computer--alpha carbon pseudo bonding as a constraint for interpolation in internal coordinate space.

A Java program has been developed to produce animations and movies of protein conformation changes. The animations, primarily intended for teaching purposes, are produced by visualizing a series of aligned structures interpolated between two forms of the same protein. To produce plausible intermediate structures, the interpolation is performed in internal coordinate space and uses a simple constraint to avoid the production of artifactual movements of the protein backbone. This constraint involves the introduction of 'pseudo' bonds linking the protein alpha carbon atoms. All of the steps from alignment of initial structures to the production of AVI movies can be performed on readily available personal computers.

Computer Simulation↗
PubMed1987

Calcium-sensitive, lipid-binding cytoskeletal proteins of the human placental microvillar region.

In this study we describe a group of Ca2+-sensitive proteins located in the microvillar region of the human placental syncytiotrophoblast. By following the distribution of proteins between the particulate and supernatant phases of detergent-solubilized microvilli in the presence of defined concentrations of free Ca2+, we demonstrate a class of proteins of subunit molecular weights 72,000, 69,000, 38,000, 36,000, and 32,000 that associate with both the cytoskeleton and lipid at high concentrations of free Ca2+. These proteins can be released from microvilli using EGTA-containing buffers. Although they do not bind to phenyl-Sepharose, they will bind to phospholipids immobilized on phenyl-Sepharose columns in a Ca2+-dependent manner and show a marked preference for phospholipids with negatively charged headgroups. The results provide evidence for a sequence of events which may occur within the microvillus as the localized concentration of intracellular free Ca2+ rises.

Calcium↗
PubMed1986

Proteins of the human placental microvillar cytoskeleton. alpha-Actinin.

The human placental syncytiotrophoblast microvilli are supported by an underlying cytoskeleton consisting mainly of actin microfilaments. The major proteins associated with the actin have Mr values of 105 000, 80 000 and 68 000. The 105 000-Mr protein is recognized by an antibody preparation raised to purified chicken gizzard alpha-actinin. Electron microscopy has shown that the human placental protein has dimensions similar to those reported for muscle alpha-actinin. About half of the placental microvillar alpha-actinin is released from the cytoskeleton in the presence of Ca2+. This effect occurs at concentrations of Ca2+ greater than 0.3 muM and has been used as the basis of a method for the purification of the placental alpha-actinin. This sensitivity to Ca2+ is not affected by trifluoperazine and is therefore likely to be a property of the alpha-actinin as such rather than being mediated via calmodulin.

Actinin↗
PubMed1985

Rabbit peroxidase-antiperoxidase complex (PAP) as a model for the uptake of immunoglobulin G by the human placenta.

Rabbit peroxidase-antiperoxidase complex (PAP) has been shown to bind to IgG receptors on the human placental syncytiotrophoblast microvillar membrane. Its binding characteristics suggest that it is suitable as a probe for studies on the uptake of IgG by the human placenta. A novel assay system was developed to measure the dissociation constants (Kd) of the binding of PAP and of unlabelled human IgG to purified placental microvillar membranes. The Kd for PAP was found to be 54 nM, while that for unlabelled IgG was found to be 17.5 nM. The uptake of PAP by placental tissue slices was observed using peroxidase histochemistry and electron microscopy. In initial experiments, reaction product was confined to the peripheral regions of the syncytiotrophoblast. Assaying a placental homogenate for catalase activity showed that it contained 250 units of activity per g wet weight of tissue (compared with 680 units/g for rat liver). Treatment of fixed tissue with the catalase inhibitor 3-amino-1, 2, 4-triazole allowed the localization of peroxidase reaction product in deeper regions of the syncytiotrophoblast. Based on observations of the localization of reaction product, we propose that PAP is taken up in coated pits, transferred into large apical multivesicular bodies, segregated into small vesicles which then transport it to the Golgi. From here the PAP is directed to the basal membrane by a mechanism as yet unknown.

Animals↗
PubMed1983

Structure of human placental microvilli.

Cytoskeletons have been prepared from microvilli isolated from the human placental syncytiotrophoblast. They contain actin and a protein similar to fimbrin. In addition, they contain calmodulin and a protein of relative molecular mass (Mr) 105 000, both of which can be released from the cytoskeletons by treatment with Ca2+. In this respect the 105 000 Mr protein is more similar to non-muscle alpha-actinin than to the intestinal microvillar protein with an Mr of 110 000. Human placental actin displays the anomalous properties of binding to phenyl-Sepharose and wheatgerm agglutinin-Sepharose, suggesting that one or more membrane glycoproteins is associated with the actin. Transferrin, presumably receptor-bound, has been identified in preparations of extensively washed placental microvillar cytoskeletons. These findings are discussed in terms of the earliest events in endocytosis and materno-fetal transfer.

Actins↗
PubMed1982

The actions of composite trophoblast antigens in microvillus preparations upon cultured maternal lymphocytes from early first pregnancies.

The effect of placental microvillus preparations (MVP) on lymphocytes in whole blood cultures obtained from mothers in the early stages of their first pregnancies was studied. Variable dilutions and variable harvesting were used to construct kinetic curves of [6-3H]-thymidine uptake per day. Significant responses (P less than 0.02 to 0.05) were observed in autologous plasma and heterologous serum with and without phytohemagglutinin. The responses showed a variability which may be explained by chance combination of similar MVP and in vivo trophoblast antigens, some with stimulatory and others with suppressor actions. The presence of a blocking antibody to a suppressor antigen in autologous plasma could also contribute.

Adolescent↗
PubMed1981

Human placental coated vesicles contain receptor-bound transferrin.

Human placental coated vesicles have been purified by a method involving sucrose-density-gradient centrifugation and treatment with wheat-germ agglutinin. These preparations were free of contamination by placental microvillus fragments. Crossed immunoelectrophoresis demonstrated that the coated vesicles contained a single serum protein, which was identified as transferrin. This transferrin was only observed after the vesicles were treated with a non-ionic detergent, and its behaviour during crossed hydrophobic-interaction immunoelectrophoresis suggested that a large proportion of it was receptor-bound. No other serum proteins, including immunoglobulin G, could be detected in these preparations. Receptor-bound transferrin was the only antigen common to placental coated vesicles and microvilli, implying that other plasma-membrane proteins are excluded from the region of membrane involved in coated-vesicle formation.

Cell Fractionation↗
PubMed1980

Proteins of the kidney microvillar membrane. Asymmetric labelling of the membrane by lactoperoxidase-catalysed radioiodination and by photolysis of 3,5-di[125I]iodo-4-azidobenzenesulphonate.

Two methods were used to label pig kidney microvillar membrane proteins from the luminal and cytoplasmic surfaces of closed membrane vesicles. The first method was lactoperoxidase-catalysed radioiodination. The enzyme reagents, lactoperoxidase and glucose oxidase, were positioned inside the vesicles before sealing or externally after sealing, iodination being initiated by the subsequent addition of glucose and 125I-. After resolution of the labelled proteins by electrophoresis in the presence of dodecyl sulphate, asymmetric labelling patterns on radioautographs were observed. However, the major disadvantage of this method is the high degree of intramembrane labelling of the fatty acid chains of membrane lipids, a reaction that undermines any conclusions about the location of the label in that region of the protein supposedly exposed at the surface of the membrane. The second method overcame this disadvantage. A new hydrophilic photoreagent, 3,5-di[125I]iodo-4-azidobenzesulphonate, was synthesized via the intermediate, diazotized 3,5-di[125I]iodosulphanilic acid. It was transported by a Na+-dependent system into microvillar vesicles, thus permitting labelling from either side of the membrane when the vesicles were photolysed. The labelling of membrane lipids was less than with the first method and was essentially confined to the polar headgroups. The activity of several microvillar peptidases survived the labelling reaction and they could be identified in the immunoprecipitates after resolution of the detergent-solubilized membrane proteins by crossed-immunoelectrophoresis. Treatment with papain converted the detergent-solubilized form of susceptible enzymes into the proteinase-solubilized form, which lacked the intramembrane domain and any portion exposed at the cytoplasmic surface. Radioautography established that aminopeptidases M and A, dipeptidyl peptidase IV and neutral endopeptidase were transmembrane proteins. This novel approach to the investigation of membrane topology may be applicable to other complex membranes.

Animals↗
PubMed1979

Proteins of the kidney microvillar membrane. Immunoelectrophoretic analysis of the membrane hydrolases: identification and resolution of the detergent- and proteinase-solubilized forms.

Antibodies raised in rabbits to detergent-solubilized pig kidney microvillar proteins have been used to investigate the membrane hydrolases by crossed immunoelectrophoresis. Eight enzymes were detected by specific staining methods: aminopeptidase M, dipeptidylpeptidase IV, neutral endopeptidase, aminopeptidase A, carboxypeptidase P, gamma-glutamyltransferase, trehalase and phosphodiesterase I. The mobility of all these enzymes, with the exception of trehalase and neutral endopeptidase, was increased by treatment of the detergent-solubilized preparation with papain. The difference between the detergent and proteinase forms of these enzymes is attributed to the removal of a small, non-antigenic peptide to which detergent is bound in significant quantities. This interpretation was further supported by experiments in which the microvillus fraction was labelled with an intramembrane photolabelling reagent, 1-azido-4-[125I]iodobenzene. After photolysis, the radioactivity in the membrane could be solubilized by detergent treatment but not by papain treatment. Radioautography after crossed charge-shift immunoelectrophoresis showed a good correlation between charge-shift (signifying the presence of detergent bound to a hydrophobic domain) and the presence of the label.

Animals↗
PubMed1977

A novel system for the two-dimensional electrophoresis of membrane proteins.

Membrane proteins were resolved in two dimensions by a novel technique that uses discontinuous electrophoresis in both directions. After electrophoresis in the first direction in chloral hydrate, the membrane proteins were further resolved by a novel system that used organic-base dodecyl sulphates to stack and then resolve them. This latter system has several advantages over conventional electrophoresis in sodium dodecyl sulphate, notably that it avoids the production of artifacts generated by other systems.

Animals↗
PubMed1977

Peptidases of the kidney microvillus membrane.

The microvillus membrane of the kidney is highly differentiated in its complement of enzymes and other proteins. In addition to the five well documented peptidases that are present in the membrane, recent work suggests that aminopeptidase P, carboxypeptidase P and an enzyme tentatively referred to as "leucine hydrazidase" are also microvillus enzymes. Microvillus serine peptidase (dipeptidyl peptidase IV) has been purified after detergent solubilization. The catalytic and molecular properties of this form and the form released by autolysis have been compared in an attempt to gain understanding of the intramembranous domain of this protein. Current views on the molecular organization of the microvillus are discussed.

Animals↗
PubMed1976

Dipeptidyl peptidase IV, a kidney brush-border serine peptidase.

Dipeptidyl peptidase IV, an enzyme that releases dipeptides from substrates with N-terminal sequences of the forms X-Pro-Y or X-Ala-Y, was purified 300-fold from pig kidney cortex. The kidney is the main source of the enzyme, where it is one of the major microvillus-membrane proteins. Several other tissues contained demonstrable activity against the usual assay substrate glycylproline 2-naphthylamide. In the small intestine this activity was greatly enriched in the microvillus fraction. In all tissues examined, the activity was extremely sensitive to inhibition by di-isopropyl phosphorofluoridate (Dip-F), but relatively resistant to inhibition by phenylmethylsulphonyl fluoride. It is a serine proteinase which may be covalently labelled with [32P]Dip-F, and is the only enzyme of this class in the microvillus membrane. The apparent subunit mol.wt. estimated by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis and by titration with [32P]Dip-F was 130 000. Gel-filtration and sedimentation-equilibrium methods gave values in the region of 280 000, which is consistent with a dimeric structure, a conclusion supported by electron micrographs of the purified enzyme. Among other well-characterized serine proteinases, this enzyme is unique in its membrane location and its large subunit size. Investigation of the mode of attack of the peptidase on oligopeptides revealed that it could hydrolyse certain N-blocked peptides, e.g. Z-Gly-Pro-Leu-Gly-Pro. In this respect it is acting as an endopeptidase and as such may merit reclassification and renaming as microvillus-membrane serine peptidase.

Animals↗