Purification and characterization of a flavin-binding storage protein from the hemolymph of Galleria mellonella.
The 85K storage protein that accumulates in the hemolymph of Galleria mellonella during the final larval instar was isolated and purified from newly molted pupae. The separation of fresh hemolymph proteins from larvae or pupae by different chromatographic and electrophoretic procedures indicated the native protein had a M(r) of 170,000 and consisted of two identical 85K subunits. Crosslinking experiments using fresh hemolymph followed by Western blotting also indicated a dimeric structure for the native protein. Analyses of the dimer purified from pupal hemolymph indicated that 85K was a glycoprotein, containing approximately 6.5% neutral sugar and about 1.9% amino sugar. Like other insect flavin-binding proteins, 85K has a relatively high histidine content but an uncharacteristically high arginine content. The purified 85K dimer did not bind riboflavin, suggesting that the integrity of the molecule had been altered during purification. However, 85K purified in low yield by Affi-Gel Blue chromatography, did bind riboflavin, indicating that under certain, undefined conditions the functional integrity of the protein could be retained during purification.
Silhacek
D L
DL
Insect Attractants, Behavior, and Basic Biology Research Laboratory, U.S. Department of Agriculture/Agricultural Research Service, Gainesville, FL 32604.
Miller
S G
SG
Murphy
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000737:chemistry*; D007814:Larva / Q000737:chemistry; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000737:chemistry*; D011679:Pupa / Q000737:chemistry; D012256:Riboflavin / Q000378:metabolism*
D002241:Carbohydrates; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein; D012256:Riboflavin