Properties and significance of a riboflavin-binding hexamerin in the hemolymph of Hyalophora cecropia.
A riboflavin-binding hexamerin isolated from pupal hemolymph of Hyalophora cecropia has a native M(r) of 510,000, subunit M(r) of 85,000, and a 5% carbohydrate content. An intrachain cross-link was confirmed in protease limit digests. Ellman titration confirmed the presence of a sulfhydryl group, which is needed for this linkage. Though Cu2+ is known to promote the linkage, heavy metals were not detected in the isolate. Heat denaturation released ligand with the absorbency, fluorescence spectra, and chromatographic behavior of riboflavin. Binding resulted in substantial quenching of the fluorescence of both the isoalloxazine in riboflavin and of aromatic groups in the apoprotein. Kinetic analysis indicated a KD of 2.5 x 10(-7) M for riboflavin, 1.3 x 10(-7) M for lumiflavin, and greater than 1 x 10(-6) M for FMN and FAD. Over four moles of flavin were bound per mole of hexamerin. The amount of riboflavin in pupal hemolymph is sufficient to occupy only 2-3 of these sites. Riboflavin is also associated with lipophorin and vitellogenin, but the molar ratios after protein isolation were low. On a standard laboratory diet, riboflavin is in great excess, but most of it is apparently excreted before the apoprotein first appears in the hemolymph, just before wandering. The concentration of riboflavin-binding hexamerin rises to 15-30 mg/ml in pupae; relative to other hexamerins, very little is stored in the fat body. All of the apoprotein and 75% of riboflavin disappear from the hemolymph during adult development. An amount of flavin at least equal to that stored in pupal hemolymph is transferred to the eggs formed during this period.
Magee
J
J
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
Kraynack
N
N
Massey
H C
HC
Telfer
W H
WH
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006458:Hemolymph / Q000378:metabolism*; D007301:Insect Hormones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019476:Insect Proteins; D007700:Kinetics; D007814:Larva / Q000378:metabolism; D008297:Male; D008317:Malpighian Tubules / Q000737:chemistry; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D009036:Moths / Q000378:metabolism*; D011679:Pupa / Q000378:metabolism; D012256:Riboflavin / Q000378:metabolism*
D002352:Carrier Proteins; D007301:Insect Hormones; D019476:Insect Proteins; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein; C041137:storage proteins, Insecta; D012256:Riboflavin