Erythrocyte membrane skeletal protein bands 4.1 a and b are sequence-related phosphoproteins.

S R Goodman,J Yu,C F Whitfield,E N Culp,E J Posnak

doi:10.1016/s0021-9258(18)34761-6

Abstract

Bands 4.1 a and b are proteins of 80,000 and 78,000 molecular weight, which are both present at approximately 100,000 copies per erythrocyte ghost. Both proteins are components of the erythrocyte membrane skeleton. Bands 4.1 a and b are labeled when intact erythrocytes are incubated with [32P]orthophosphoric acid, and, therefore, are phosphoproteins. One-dimensional partial proteolytic mapping analysis of 32P-labeled bands 4.1 a and 4.1 b and two-dimensional peptide mapping analysis of 125I-labeled bands 4.1 a and 4.1 b clearly demonstrated that the two proteins are sequence-related phosphoproteins. Band 4.1 purified by standard techniques (Tyler, J. M., Hargreaves, W. R., and Branton, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5192-5196) contains bands 4.1 a and 4.1 b. Bands 4.1 a and 4.1 b bind to spectrin heterodimers in solution. We conclude that the erythrocyte skeletal proteins bands 4.1 a and 4.1 b are sequence-related phosphoproteins, both capable of binding spectrin.

Highlights

Molecular weight, which are both present at -100,000 Since spectrin tetramers are formed by head-to-head associcopies per erythrocyte ghost
Preparation of '"P-labeled Erythrocyte Ghosts-Erythrocyte membrane phosphoproteins were ''*P-labeled by metabolically labeling intact erythrocytes (6 ml of packed cells) with ["Plorthophosphoric acid (10 mCi) by the method of Bennett and Branton[29].'"Plabeled erythrocyteghosts were prepared in the Same manner as unlabeled ghosts
Preparation of [32PJSpectrin Heterodimer-"P-labeled erythrocyte ghosts were washed in 0.3 mM NaPOn,0.2 mM EDTA, pH 7.6

Summary

EXPERIMENTAL PROCEDURES

Materials ["P]Orthophosphoric acid (20 mCi/ml in 0.02 N HCI) and monoiodinated '*'I-labeled Bolton-Hunter reagent (2000 Ci/mmol) were from New England Nuclear. '*'I (-17 mCi/mmol) was from Amersham Corp. Materials ["P]Orthophosphoric acid (20 mCi/ml in 0.02 N HCI) and monoiodinated '*'I-labeled Bolton-Hunter reagent (2000 Ci/mmol) were from New England Nuclear. '*'I (-17 mCi/mmol) was from Amersham Corp. DFP, Coomassie brilliant blue (R),diphenyl carbamyl chloride-treated trypsin, a chymotrypsin, and thermolysin were from Sigma. Acrylamide, N,N'-methylenebisacryylamide, ammonium persulfate, TEMED, and SDS were from Bio-Rad

Methods

RESULTS

DISCUSSION