Biochemical studies on an oocyte membrane receptor for the biliprotein, insecticyanin, of the hawkmoth Manduca sexta

Persistent Link:
http://hdl.handle.net/10150/282178
Title:
Biochemical studies on an oocyte membrane receptor for the biliprotein, insecticyanin, of the hawkmoth Manduca sexta
Author:
Kang, Yang, 1957-
Issue Date:
1996
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Sequestration of the blue biliprotein, insecticyanin, into developing oocytes of the hawkmoth, Manduca sexta was investigated using diverse techniques. Oudin's immunodiffusion assays revealed that insecticyanin concentration in mature eggs (29.6 μM) is slightly higher than that in hemolymph (25.8 μM). The endocytotic uptake of insecticyanin was visualized at the light microscopic level using autoradiography. Uptake of ¹²⁵I-insecticyanin by isolated oocytes was ligand specific, saturable with increasing insecticyanin concentration, and sensitive to parameters such as size of oocyte, constituents of media and temperature. Analysis of in vitro uptake data yielded values of K(uptake) (insecticyanin concentration at half-maximal uptake rate) of 4.2 μM and V(max) (maximum rate of uptake) of 1 pmol follicle⁻¹ h⁻¹. Oocyte membrane proteins were efficiently solubilized using 40 mM detergent CHAPS. Labeled insecticyanin was shown to bind to crude follicle membranes and solubilized membrane proteins with high specificity and affinity. The K(d) (equilibrium dissociation constant) was estimated as 40 nM and 17 nM for crude membranes and solubilized membrane proteins, respectively. The B(m) (maximum binding) estimated from crude membrane and solubilized membrane protein was 1.6 and 11.4 pmol/mg proteins respectively. Competition studies showed that binding of labeled insecticyanin to its receptor was blocked by an excess of unlabeled insecticyanin but not by other major hemolymph proteins, lipophorin and vitellogenin of M. sexta. Additional binding experiments demonstrated that receptors for insecticyanin are only present in oocyte membranes, not in fat body, gut tissue or ovariole. The results from co-immunoprecipitation showed that the apparent molecular mass for insecticyanin receptor is approximately 185 kDa on reducing SDS-PAGE gel while chemical crosslinking of the insecticyanin-receptor complex revealed a product with a molecular mass near 1000 kDa. This result suggests that the insecticyanin receptor has a multimeric structure, or that four receptor molecules can bind to one insecticyanin tetramer.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.; Biology, Entomology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Law, John H.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleBiochemical studies on an oocyte membrane receptor for the biliprotein, insecticyanin, of the hawkmoth Manduca sextaen_US
dc.creatorKang, Yang, 1957-en_US
dc.contributor.authorKang, Yang, 1957-en_US
dc.date.issued1996en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractSequestration of the blue biliprotein, insecticyanin, into developing oocytes of the hawkmoth, Manduca sexta was investigated using diverse techniques. Oudin's immunodiffusion assays revealed that insecticyanin concentration in mature eggs (29.6 μM) is slightly higher than that in hemolymph (25.8 μM). The endocytotic uptake of insecticyanin was visualized at the light microscopic level using autoradiography. Uptake of ¹²⁵I-insecticyanin by isolated oocytes was ligand specific, saturable with increasing insecticyanin concentration, and sensitive to parameters such as size of oocyte, constituents of media and temperature. Analysis of in vitro uptake data yielded values of K(uptake) (insecticyanin concentration at half-maximal uptake rate) of 4.2 μM and V(max) (maximum rate of uptake) of 1 pmol follicle⁻¹ h⁻¹. Oocyte membrane proteins were efficiently solubilized using 40 mM detergent CHAPS. Labeled insecticyanin was shown to bind to crude follicle membranes and solubilized membrane proteins with high specificity and affinity. The K(d) (equilibrium dissociation constant) was estimated as 40 nM and 17 nM for crude membranes and solubilized membrane proteins, respectively. The B(m) (maximum binding) estimated from crude membrane and solubilized membrane protein was 1.6 and 11.4 pmol/mg proteins respectively. Competition studies showed that binding of labeled insecticyanin to its receptor was blocked by an excess of unlabeled insecticyanin but not by other major hemolymph proteins, lipophorin and vitellogenin of M. sexta. Additional binding experiments demonstrated that receptors for insecticyanin are only present in oocyte membranes, not in fat body, gut tissue or ovariole. The results from co-immunoprecipitation showed that the apparent molecular mass for insecticyanin receptor is approximately 185 kDa on reducing SDS-PAGE gel while chemical crosslinking of the insecticyanin-receptor complex revealed a product with a molecular mass near 1000 kDa. This result suggests that the insecticyanin receptor has a multimeric structure, or that four receptor molecules can bind to one insecticyanin tetramer.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
dc.subjectBiology, Entomology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLaw, John H.en_US
dc.identifier.proquest9713424en_US
dc.identifier.bibrecord.b34434860en_US
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