Separation and Characterization of the Subunits of the Laminin of EHS Sarcoma

Arumugham, Rasappa; Trumbore, Mark W.; Thomas, T. Anto; Makhlouf, S.; Tanzer, Marvin L.

doi:10.3109/03008208809008065

Cited by 5 publications

(8 citation statements)

References 35 publications

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“…We have used gold-labelled heparin-albumin as a specific probe for the laminin A subunit on Western blots and have shown that the heparin-binding site on the laminin A subunit must reside on the polypeptide backbone and not on carbohydrate side chains, since the probe bound to the deglycosylated A subunit. Several heparin-binding sites have been identified on EHS laminin (Ott et al, 1982;Skubitz et al, 1988) and localized to the A subunit (Arumugham et al, 1988) and to the B1 subunit (Charonis et al, 1988). Our results suggest, in agreement with Arumugham et al (1988), that only the heparin-binding domain on the A subunit is detectable after the reduction and denaturation of laminin.…”

Section: Discussionsupporting

confidence: 86%

“…Several heparin-binding sites have been identified on EHS laminin (Ott et al, 1982;Skubitz et al, 1988) and localized to the A subunit (Arumugham et al, 1988) and to the B1 subunit (Charonis et al, 1988). Our results suggest, in agreement with Arumugham et al (1988), that only the heparin-binding domain on the A subunit is detectable after the reduction and denaturation of laminin.…”

Section: Discussionsupporting

confidence: 86%

“…To confirm that the major protein band at 400 kDa had been correctly identified as the laminin A subunit, electroblots of deglycosylated murine EHS laminin were probed with gold-labelled heparin-albumin. Heparin has been shown to bind to the A subunit, but not the B subunits, of reduced and alkylated EHS laminin (Arumugham et al, 1988). As the results in Fig.…”

Section: Deglycosylation Of Murine Ehs Lamininmentioning

confidence: 58%

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Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-β-galactosidase fusion proteins

Brown¹,

Spragg²,

Wheeler³

et al. 1990

Biochemical Journal

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Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionsupporting

confidence: 86%

Section: Deglycosylation Of Murine Ehs Lamininmentioning

confidence: 58%

See 1 more Smart Citation

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-β-galactosidase fusion proteins

Brown¹,

Spragg²,

Wheeler³

et al. 1990

Biochemical Journal

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“…Western blots were carried out typically in 9% acrylamide gels using the Laemmli gel system and were electroblotted to nitrocellulose (20). RPN800 Rainbow marker (Amersham Pharmacia Biotech) and laminin (subunits 200 and 400 kDa; GIBCO) were used as molecular weight markers (4). The primary antibodies used were 5F10 (mouse monoclonal anti-PMCA pump antibody; Affinity BioReagents, Golden, CO) and 4G5 (mouse monoclonal anti-SERCA pump antibody; gift from Dr. L. R. Jones, Univ.…”

Section: Methodsmentioning

confidence: 99%

Effects of peroxynitrite on sarcoplasmic reticulum Ca²⁺ pump in pig coronary artery smooth muscle

Grover

Samson²,

Robinson³

et al. 2003

American Journal of Physiology-Cell Physiology

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Peroxynitrite generated in arteries from superoxide and NO may damage Ca(2+) pumps. Here, we report the effects of peroxynitrite on ATP-dependent azide-insensitive uptake of Ca(2+) into pig coronary artery vesicular membrane fractions F2 [enriched in plasma membrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)]. Membranes were pretreated with peroxynitrite and then with DTT to quench this agent. This pretreatment inhibited Ca(2+) uptake in a peroxynitrite concentration-dependent manner, but the effect was more severe in F3 than in F2. The inhibition was thus not overcome by excess DTT used to quench peroxynitrite and was not affected if catalase, SOD, or mannitol was added along with peroxynitrite. Such damage to the pump protein would be difficult to repair if produced during ischemia-reperfusion. The acylphosphates formed with ATP in F3 corresponded mainly to the SR Ca(2+) pump (110 kDa), but in F2 both PM (140 kDa) and 110-kDa bands were observed. Peroxynitrite treatment of F2 inhibited only the 110-kDa band. Inhibition of Ca(2+) uptake and acylphosphate formation from ATP correlated well in peroxynitrite-treated F3 samples. However, inhibition of acylphosphates from orthophosphate (reverse reaction of the pump) was slightly poorer. Peroxynitrite treatment also covalently cross-linked the pump protein, yielding no dimers but only larger oligomers. In contrast, cross-linking of the SR Ca(2+) pump in skeletal and cardiac muscles gives dimers as the first oligomers. Therefore, we speculate that SERCA2 has a different quaternary structure in the coronary artery smooth muscle.

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“…Indeed, heparin-agarose chromatography has been routinely employed (24) to separate laminin-1 subunits. Despite reports suggesting a strong binding between laminin-1 and heparin or heparan sulfate, to our knowledge, this is the first report of a specific extended oligo- saccharide sequence in HS that has high affinity for laminin-1.…”

Section: Discussionmentioning

confidence: 99%

Oligosaccharide Sequence of Human Breast Cancer Cell Heparan Sulfate with High Affinity for Laminin

Parthasarathy

Gotow²,

Bottoms³

et al. 1998

Journal of Biological Chemistry

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Separation and Characterization of the Subunits of the Laminin of EHS Sarcoma

Cited by 5 publications

References 35 publications

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-β-galactosidase fusion proteins

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-β-galactosidase fusion proteins

Effects of peroxynitrite on sarcoplasmic reticulum Ca²⁺ pump in pig coronary artery smooth muscle

Oligosaccharide Sequence of Human Breast Cancer Cell Heparan Sulfate with High Affinity for Laminin

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Separation and Characterization of the Subunits of the Laminin of EHS Sarcoma

Cited by 5 publications

References 35 publications

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-β-galactosidase fusion proteins

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-β-galactosidase fusion proteins

Effects of peroxynitrite on sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle

Oligosaccharide Sequence of Human Breast Cancer Cell Heparan Sulfate with High Affinity for Laminin

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Effects of peroxynitrite on sarcoplasmic reticulum Ca²⁺ pump in pig coronary artery smooth muscle