Year-end Sale % OFF 
Abstract
Monoclonal antibodies were utilized to localize novel heparin-binding domains of laminin. A solid-phase radioligand binding assay was designed such that [3H] heparin bound to laminin in a time- and concentration-dependent manner. Tritiated heparin binding to laminin was saturable and specific as determined by competition with unlabeled heparin, dextran sulfate, and dermatan sulfate. By Scatchard analysis, two distinct dissociation constants were calculated (Kd = 50 and 130 nM), suggesting the presence of at least two binding sites for heparin on laminin. Tritiated heparin bound to thrombin-resistant (600 kDa) and chymotrypsin-resistant (440 kDa) laminin fragments, both known to lack the terminal globular domain of the long arm. Sodium dodecyl sulfate-polyacrylamide gels of chymotrypsin- and thermolysin-digested laminin chromatographed on a heparin-Sepharose column showed multiple proteolytic fragments binding to the column. Monoclonal antibodies generated against laminin were tested for their ability to inhibit [3H]heparin binding to laminin. Four monoclonal antibodies significantly inhibited the binding of [3H]heparin to laminin in the range of 15-21% inhibition. Laminin-monoclonal antibody interactions examined by electron microscopy showed that one antibody reacted at the terminal globular domain of the long arm, domain Hep-1, while epitopes for two of these monoclonal antibodies were located on the lateral arms of laminin, domain Hep-2, and the fourth monoclonal antibody bound below the cross-region of laminin, domain Hep-3. When two monoclonal antibodies recognizing distinctly different regions of laminin were added concomitantly, the inhibition of [3H]heparin binding to laminin increased almost 2-fold. These results suggest that at least two novel heparin-binding domains of laminin may be located in domains distinct from the terminal globular domain of the long arm.
Highlights
Monoclonal antibodies were utilized to localize novel cated in domains distinct from the terminal globular heparin-binding domains of laminin
More recent studies have used proteolytic fragments of Monoclonal antibodies generated against laminin werleaminin in conjunction with heparin-affinity chromatography tested for their ability to inhibit [‘Hlheparin binding to localize a heparin-binding domain on to laminin
Ular domain of the long arm, domain Hep-1, while In this study, we searched for additional heparin-binding epitopes for two of these monoclonal antibodies were sites on laminin by use of heparin-affinity chromatography located on the lateral armsof laminin, domain Hep-2, and a solid-phase radioligand binding assay (RLBA).l We and the fourth monoclonal antibody bound below the used laminin fragments produced aftershort-term limited cross-region of laminin, domain Hep-3
Summary
Time Courseof PHIHeparin Binding to Various Concentrations oflaminin-The binding of [3H]heparinto laminin was measured by a direct solid-phase RLBA as described under "Materials and Methods" (Fig. 1).Binding of [3H]heparin to laminin increased as a function of time, and was detected as early as 5 min when high concentrations of laminin were used. Significant binding of [3H]heparinwas achieved when wells contained greater than 0.44 pmol of laminin, and the binding of [3H]heparinincreased as theamount of laminin added was increased. Three hours was shown to be more than enough time to allow complete binding of [3H]heparin to laminin (Fig. 1).The results showed that [3H]heparin binding to laminin was saturable (Fig. 2). Reversibility of PHIHeparin Binding to Laminin-A competition assay was conducted to determine if heparin binding to laminin was reversible. Inthis assay, [3H]heparin was bound to laminin for 2 h, and a 100-fold excess of unlabeled heparin was added (Fig. 3).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
