Effects of pronase and concanavalin A upon the freeze-etch morphology of cell membranes of intact human erythrocytes

1978 ◽  
Vol 30 (1) ◽  
pp. 63-76
Author(s):  
G.E. Wise ◽  
F.L. Shienvold ◽  
R.W. Rubin
Keyword(s):  
Plasma Membrane ◽  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Particle Distribution ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Erythrocytes ◽  
Sodium Dodecylsulphate ◽  
Con A ◽  
Erythrocyte Plasma Membrane ◽  
Pronase Treatment

Freeze-etch studies reveal that mild pronase treatment with subsequent incubation in concanavalin A induces aggregation of intramembranous particles (IMP) in intact human erythrocytes. This alteration in particle distribution is accompanied by a change in the distribution of the Con A molecules such that they also become clustered on the extracellular etch face. If divalent succinyl Con A is used after pronase instead of tetravalent Con A the IMPs still become clustered. Pronase only, Con A only, or succinyl Con A only does not cause the IMPs to become aggregated. Most surprising is the finding that pronase followed by Con A causes partial haemolysis of the cells whereas pronase only, Con A only, or pronase+succinyl Con A do not cause this haemoglobin loss. These perturbations of the erythrocyte plasma membrane appear to be a result of the pronase+Con A exerting a transmembrane effect on the spectrin. This conclusion is supported by sodium dodecylsulphate polyacrylamide gel electrophoresis of material crosslinked with dimethyl adipimidate dihydrochloride, which indicates that spectrin is more susceptible to being cross-linked after pronase+Con A; i.e. the spectrin is probably aggregated by the enzyme and lectin incubation.

scholarly journals A study of maturation events in jackbeans (Canavalia ensiformis)

1984 ◽  
Vol 222 (1) ◽  
pp. 265-268 ◽  
Author(s):  
S E Marcus ◽  
J Burgess ◽  
P R Maycox ◽  
D J Bowles
Keyword(s):  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Molecular Forms ◽  
Con A ◽  
Canavalia Ensiformis ◽  
Immature Seeds

Maturation events have been studied in developing jackbean (Canavalia ensiformis) cotyledons by using a combination of analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, overlays with 125I-concanavalin A (Con A) and the use of anti-(Con A) after Western transfer. The number of polypeptides recognized by 125I-Con A varies during maturation, until at maturity only one remains. Several molecular forms of the lectin occur during development; one, corresponding to Mr 33 000 and found only in immature seeds, interacts with 125I-Con A, suggesting that it is glycosylated.

scholarly journals Surface glycoproteins of human spermatozoa

1986 ◽  
Vol 82 (1) ◽  
pp. 11-22
Author(s):  
M. Kallajoki ◽  
I. Virtanen ◽  
J. Suominen
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Spermatozoa ◽  
Galactose Oxidase ◽  
Sperm Cells ◽  
Neuraminidase Treatment ◽  
Triton X

The surface membrane glycoprotein composition of human spermatozoa has been studied by introducing radioactive label into galactosyl (Gal) and N-acetylgalactosaminyl (GalNAc) residues by using the galactose oxidase/NaB3H4 method. Triton X-100 extracts and Triton X-100-resistant cytoskeletal residues were subjected to analysis by polyacrylamide gel electrophoresis. The distribution of the radiolabel in sperm cells was studied by light-microscopic auto-radiography. The grains were evenly distributed on the cells by the labelling methods used. The Triton X-100 treatment did not affect sperm morphology at the light-microscopic level, but in transmission electron microscopy the plasma membrane covering the acrosome was removed totally, together with most of the acrosomal membranes and acrosomal contents. Plasma membrane residues were, however, always found in the postacrosomal region. Borohydride alone without oxidative pretreatment labelled two polypeptides of molecular weights (Mr) 48,000 and 43,000 in the Triton X-100-soluble fraction. When the Gal/GalNAc residues were labelled by galactose oxidase pretreatment 120,000, 105,000, 78,000 and 68,000 Mr glycoproteins were revealed. When additional neuraminidase treatment was used to remove terminal sialic acid residues, the total labelling intensity was increased two- to fivefold and additional 36,000 and 20,000 Mr glycoproteins were revealed. The Triton X-100-resistant cytoskeletal residue contained 53–75% of the total radioactivity bound in sperm cells. When these components were analysed by polyacrylamide gel electrophoresis, all the major bands found in the Triton X-100-soluble fraction were detected and also some radioactivity was incorporated into the major bands visualized by protein staining. In the present study we describe several human sperm glycoproteins, which seem to be distributed evenly on the sperm cells. Detergent extraction, producing cytoskeletal models, appeared to leave most of the glycoproteins detectable in the extraction residues also with the apparent enrichment of a single 68,000 Mr glycoprotein.

scholarly journals Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield

1981 ◽  
Vol 197 (2) ◽  
pp. 519-522 ◽  
Author(s):  
E G Afting ◽  
M L Recker
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Acid Proteinase ◽  
Chromatographic Purification

Cathepsin D was purified by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin-Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.

Studies of the human testis. XIX

1985 ◽  
Vol 108 (2) ◽  
pp. 284-288
Author(s):  
Abdul Waheed ◽  
Stephen J. Winters ◽  
George M. Farrow ◽  
Hiroyuki Oshima ◽  
Philip Troen
Keyword(s):  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Gel Filtration ◽  
Human Testis ◽  
Ammonium Sulphate Precipitation ◽  
Titration Curves ◽  
Androgen Binding Protein ◽  
Androgen Binding

Abstract. Human testosterone-oestradiol-binding globulin (hTeBG) was purified from pregnancy serum by ammonium sulphate precipitation, preparative flatbed electrofocussing, Concanavalin A-Sepharose affinity chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex chromatography and preparative polyacrylamide gel electrophoresis. The yield was 0.3 mg of hTeBG with a specific acitivity of 1.1 nmoles DHT bound per mg. An antiserum to TeBG was raised in rabbits. Anti-hTeBG IgG was separated from rabbit TeBG by DEAE-Affi-Gel-Blue chromatography. Anti-hTeBG was titrated using protein A-Sepharose which quantitatively binds IgG and therefore bound [3H]DHT-hTeBG-anti-TeBG complexes. The androgen binding components from human testis were separated on Concanavalin A-Sepharose columns into excluded and retained fractions. The antibody bound both testis fractions with titration curves which paralleled that of TeBG, indicating that these androphilic proteins share common immunodeterminants with hTe-BG. The possibility that these testicular proteins are identical in amino acid sequence to TeBG and differ only in carbohydrate content will require further verification. Finally, these results indicate that antibodies to TeBG can be used to study human testicular androgen-binding protein.

scholarly journals The distinguishing characteristics of the plasma membrane are its exposed proteins

1975 ◽  
Vol 147 (2) ◽  
pp. 373-376 ◽  
Author(s):  
R E Gates ◽  
D R Phillips ◽  
M Morrison
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Types ◽  
Distinguishing Characteristic ◽  
Probe System ◽  
Normal Human

The exposed proteins of the plasma membrane of normal human lymphocytes and platelets were labelled by using the lactoperoxidase macromolecular probe system. The labelled components were separated into molecular-weight classes by sodium dodecyl sulphate--polyacrylamide-gel electrophoresis. In contrast with the report by Tanner et al. (1974), a comparison of the two cell types showed that the major labelled components in both cell types were glycoproteins and were not identical. It is concluded that the exposed proteins are probably the most distinguishing characteristic of the plasma membrane of differentiated cell types.

scholarly journals Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes

1984 ◽  
Vol 222 (1) ◽  
pp. 235-246 ◽  
Author(s):  
N Hack ◽  
N Crawford
Keyword(s):  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Actin Binding ◽  
Good Resolution ◽  
Intracellular Membrane ◽  
Neuraminidase Treatment ◽  
Platelet Surface

By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.

The antigenicity of myosin and C-protein

1976 ◽  
Vol 192 (1109) ◽  
pp. 439-449 ◽  
Keyword(s):  
Ionic Strength ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulphate ◽  
C Protein ◽  
The Past ◽  
Homologous Antiserum ◽  
Conventional Manner ◽  
Low Ionic Strength ◽  
Repeated Precipitation

Rabbit myosin prepared in the conventional manner by repeated precipitation at low ionic strength was recently shown to contain substantial amounts of impurities; the principal impurity is a component of the myofibril called C-protein. Because antiserum to such conventionally prepared myosin has been used in the past for labelling studies of muscle, it was necessary to study the immunological characteristics of myosin and C-protein and in particular to test the specificity of this antiserum. Antisera to both rabbit myosin and C-protein have been successfully elicited in goats. These antisera have been analysed by immunodiffusion and by precipitin reactions in solution. The analysis has been helped by the examination of immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. It is concluded that: ( a ) C-protein and myosin are antigenically distinct and therefore that C-protein is not derived from myosin. ( b ) Purified myosin can behave as a classically simple antigen giving a single precipitin line when diffused against its homologous antiserum. ( c ) C-protein is a powerful immunogen; the amount present as an impurity in myosin prepared in the conventional way by repeated precipitation at low ionic strength is capable of eliciting a large amount of antibody. Consequently the pattern obtained by labelling myofibrils with antiserum to conventionally prepared myosin would contain information about the location of C-protein superimposed on information about the location of myosin.

Plasma Membrane and Nuclear Proteins of the Goose Erythrocyte

1973 ◽  
Vol 51 (10) ◽  
pp. 1442-1447 ◽  
Author(s):  
Keith R. Shelton
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nuclear Proteins ◽  
Nuclear Fraction ◽  
Nonhistone Protein ◽  
Large Molecular Weight ◽  
Goose Erythrocyte

Plasma membrane and nuclear fractions have been prepared from mature goose erythrocytes. Examination of the nonhistone protein of the nuclear fraction by sodium dodecyl sulphate – polyacrylamide gel electrophoresis reveals a limited number of molecular weight species some of which are peculiar to the nuclear fraction.Electrophoretic comparison of the goose plasma membrane proteins with those of the human erythrocyte reveals many similarities. In particular, three large molecular weight species occur in both cells. Their function appears to predate the evolutionary loss of the nucleus.

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