scholarly journals Assignment of laminin heavy chains using the lectin Ricinus communis agglutinin-1

1993 ◽  
Vol 295 (2) ◽  
pp. 537-541 ◽  
Author(s):  
J D F Wadsworth ◽  
A Okuno ◽  
P N Strong
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Ricinus Communis ◽  
Lectin Affinity Chromatography ◽  
Native Form ◽  
Heavy Chains ◽  
Lectin Affinity ◽  
Ricinus Communis Agglutinin ◽  
A Chain ◽  
Binding Glycoprotein

Using high-resolution PAGE and Western-blotting techniques the lectin Ricinus communis agglutinin-1 (RCA-1) was tested for its ability to recognize laminin subunits from the mouse Engelbreth-Holm-Swarm (EHS) tumour and from bovine cardiac and skeletal muscle. Biotinylated RCA-1 recognized both the A and B chains of purified EHS-tumour laminin with a sensitivity comparable to anti-(EHS laminin) antibodies. In cardiac and skeletal muscle RCA-1 also recognized the B chains of laminin, together with a approximately 330 kDa RCA-1-binding glycoprotein that was undetectable in smooth muscle. This glycoprotein was not recognized by antibodies raised to laminin from the EHS tumour. Purification of the 330 kDa binding glycoprotein from skeletal muscle, using ion-exchange and lectin-affinity chromatography, revealed that in its native form, this glycoprotein is disulphide-bonded to the B chains of laminin. The demonstrated properties of the approximately 330 kDa RCA-1-binding glycoprotein are identical to those reported for the variant M chain of merosin which is known to replace the A chain in laminin from the extrasynaptic regions of skeletal muscle. These results establish that biotinylated RCA-1 can recognize A-, B- and M-chain subunits of laminin isoforms, and that, when used in conjunction with other techniques, they provide a useful method for the assignment of laminin heavy chains.

scholarly journals Comparison of desialylation of rat transferrin by cellular and non-cellular methods

1989 ◽  
Vol 259 (2) ◽  
pp. 427-431 ◽  
Author(s):  
S Irie ◽  
J J Minguell ◽  
M Tavassoli
Keyword(s):  
Affinity Chromatography ◽  
Acid Hydrolysis ◽  
Ricinus Communis ◽  
Lectin Affinity Chromatography ◽  
Neuraminidase Treatment ◽  
Functional Studies ◽  
Lectin Affinity ◽  
Ricinus Communis Agglutinin ◽  
Protein Moiety

We have previously shown that the liver endothelium can desialylate the glycoprotein transferrin (Tf). In the present work we provide evidence that asialotransferrin obtained by this means behaves differently on Ricinus communis agglutinin (RCA120) lectin affinity chromatography from asialotransferrin obtained by either neuraminidase treatment or acid hydrolysis. Purified rat transferrin was radiolabelled either with 125I (protein moiety) or with 3H (sialyl residues), and subsequently saturated with iron. It was then passed through an RCA120-agarose column to isolate the fully sialylated component. Sialylated Tf was then desialylated either by incubation with purified rat liver endothelium or, in vitro, by neuraminidase treatment or by acid hydrolysis. The protein was again subjected to RCA120 column chromatography. Although both neuraminidase treatment and acid hydrolysis almost completely desialylated the glycoprotein (as evidenced by near absence of 3H label), the glycoprotein was not retained by the RCA120-agarose column. By contrast, liver endothelium partially desialylated the glycoprotein, but this desialylated fraction was retained by the RCA120-agarose column. These results suggest that desialylation with neuraminidase or acid hydrolysis may be inadequate for functional studies of asialotransferrin.

scholarly journals Investigation of different molecular forms of IGFBP-1 using immobilised metal-, immuno- and lectin-affinity chromatography

2010 ◽  
Vol 75 (11) ◽  
pp. 1481-1489 ◽  
Author(s):  
Dragana Lagundzin ◽  
Romana Masnikosa ◽  
Goran Miljus ◽  
Dragana Robajac ◽  
Olgica Nedic
Keyword(s):  
Affinity Chromatography ◽  
Great Influence ◽  
Molecular Forms ◽  
Post Translational Modification ◽  
Lectin Affinity Chromatography ◽  
Homologous Proteins ◽  
Native Form ◽  
Lectin Affinity ◽  
Reducing Conditions ◽  
Insulin Like Growth Factors

Insulin-like growth factor-binding protein 1 (IGFBP-1) is a member of a family of six homologous proteins that regulate the action of the insulin-like growth factors. IGFBP-1 is a 25 kDa protein that beside its native form, may exist in several phosphoforms (30 kDa), which are predominant in the circulation of humans. Phosphorylation of IGFBP-1 is a post-translational modification that has a great influence on the IGF-I action. IGFBP-1 forms multimers and complexes with ?2-macroglobulin (?2M). Polymerisation of IGFBP-1 was also reported. In order to analyse and separate these IGFBP-1 molecular species, affinity chromatography methods were used in this study. The results demonstrated that most of the IGFBP-1 circulates in complexes with ?2M, which can be isolated by affinity chromatography using immobilised anti-?2M antibodies. IGFBP- 1/?2M complexes may be differentiated from IGFBP-1 dimer and multimers using lectin-affinity chromatography, since the latter do not interact with lectins. It seems that the complexes contain not only monomeric IGFBP-1, but also its multimers. Dimer and multimers are stable under reducing conditions, suggesting covalent linkage between units. Free IGFBP-1 monomer can be separated from multimers using Con A-affinity chromatography. The concentration of free IGFBP-1 is relatively low in the circulation.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

Glycoprotein Ib Has a Partial Role in Platelet-von Willebrand Factor Collagen Interaction

1988 ◽  
Vol 60 (02) ◽  
pp. 182-187 ◽  
Author(s):  
Morio Aihara ◽  
Ken Tamura ◽  
Ryuko Kawarada ◽  
Keizou Okawa ◽  
Yutaka Yoshida
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Ricinus Communis ◽  
Protein S ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Normal Plasma ◽  
Ricinus Communis Agglutinin ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe adhesion of human fixed washed platelets (FWP) to collagen was decreased after treatment with Serratia marcescens protease (SP), which removed 95% of the glycocalicin from platelet membrane glycoprotein (GP) lb. However, the diminished adhesion of SP treated FWP to collagen could still be increased in the presence of purified von Willebrand factor (vWF). This ability of vWF to increase FWP adhesion to collagen is defined as collagen cofactor (CCo). The adhesion of FWP to collagen was not affected by a monoclonal antibody (MAb) to GP Ilb/IIIa (10E5), that inhibits ADP and collagen induced platelet aggregation. On the other hand, it was decreased by 50% by a MAb to GP lb (6D1), that inhibits ristocetin induced platelet aggregation. Adhesion of FWP in buffer to collagen was completely inhibited by Ricinus communis agglutinin I or concanavalin A, while Lens culinalis agglutinin and wheat germ agglutinin showed 50% inhibition. The FWP adhesion to collagen in the presence of vWF (normal plasma) was unaffected by MAbs to GP Ilb/IIIa (10E5, P2, HPL1) but was decreased to 32-38% by MAbs to GP lb (6D1, AN51, HPL11). A MAb to vWF (CLB-RAg 35), that inhibits ristocetin induced binding of vWF to platelets, decreased the CCo of normal plasma by 70%. The MAb, CLB-RAg 201, that inhibits the binding of vWF to collagen, completely inhibited the CCo of normal plasma. In conclusion, our data suggest that (1) GP lb has a partial role in FWP adhesion to collagen; (2) the binding of vWF to collagen is required for the expression of CCo; (3) CCo is partly mediated through GP lb; but (4) other platelet membrane protein(s) besides GP lb or GP Ilb/IIIa must also be involved in FWP-vWF-collagen interactions.

Further Studies on Aggregation of Platelet-Type von Willebrand’s Disease Platelets by Human von Willebrand Factor

1986 ◽  
Vol 55 (03) ◽  
pp. 338-341 ◽  
Author(s):  
H Takahashi ◽  
W Tatewaki ◽  
M Hanano ◽  
R Nagayama ◽  
A Shibata
Keyword(s):  
Von Willebrand Factor ◽  
Ricinus Communis ◽  
Divalent Cations ◽  
Peanut Agglutinin ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Ricinus Communis Agglutinin ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

SummaryPlatelet-type von Willebrand’s disease (vWD) is a bleeding disorder characterized by a heightened interaction between platelets and von Willebrand factor (vWF) as the result of an intrinsic platelet abnormality (probably in GPIb). Platelet aggregability was nearly normal in response to thrombin, wheat germ agglutinin and Ricinus communis agglutinin in this disorder. Unmodified platelets showed no aggregation upon the addition of peanut agglutinin. Partially purified human vWF induced little aggregation of washed patient platelets, but the aggregation was greatly enhanced in the presence of plasma devoid of vWF. Monoclonal antibodies directed against GPIb and GPIIb/IIIa as well as EDTA completely inhibited vWF-induced aggregation. These results indicate that human vWF induces aggregation of platelet-type vWD platelets in the presence of divalent cations and some plasma cofactor(s), and that both GPIb and GPIIb/IIIa are involved in this aggregation.

scholarly journals Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle

2000 ◽  
Vol 279 (5) ◽  
pp. C1656-C1664 ◽  
Author(s):  
B. Paul Herring ◽  
Shelley Dixon ◽  
Patricia J. Gallagher
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Cardiac Myocyte ◽  
Cardiac Tissue ◽  
Specific Protein ◽  
Myoblast Differentiation ◽  
Adult Skeletal Muscle

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or “nonmuscle” form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.

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