Ramjeesingh M, Rothstein A
Membr Biochem. 1982;4(4):259-69. doi: 10.3109/09687688209065435.
A 17,000-dalton transmembrane segment of band 3 protein is further cleaved by chymotrypsin treatment of red blood cell ghosts to 15,000 daltons. The location of this particular chymotrypsin cleavage site was determined by comparing the fragmentation pattern of the 17,000- and 15,000-dalton peptides using cyanogen bromide (CNBr). Each peptide is cleaved at its two methionine residues into three fragments. For each peptide two of the fragments are the same size, 7000 and 4000 daltons, the latter containing, in each case, the binding site of the anion transport inhibitor 4,4'-diisothiocyano-2,2' disulfonic acid (DIDS). The third fragment is 2000 daltons larger in the case of the 17,000-dalton peptide (6000 compared to 4000 daltons). These findings indicate that the chymotrypsin cleavage site is located at the cytoplasmic side of the membrane, 2000 daltons from the N-terminus of the 17,000-dalton peptide. This information allows the mapping of a number of defined sites of the 15,000-dalton segment within the primary structure of band 3. These sites support the suggestion that this peptide segment is folded within the bilayer.
用胰凝乳蛋白酶处理红细胞血影后，带3蛋白的一个17000道尔顿的跨膜片段会进一步裂解为15000道尔顿。通过使用溴化氰（CNBr）比较17000道尔顿和15000道尔顿肽段的片段化模式，确定了这个特定胰凝乳蛋白酶裂解位点的位置。每个肽段在其两个甲硫氨酸残基处裂解为三个片段。对于每个肽段，其中两个片段大小相同，为7000道尔顿和4000道尔顿，后者在每种情况下都包含阴离子转运抑制剂4,4'-二异硫氰酸-2,2'-二磺酸（DIDS）的结合位点。在17000道尔顿肽段的情况下，第三个片段大2000道尔顿（分别为6000道尔顿和4000道尔顿）。这些发现表明，胰凝乳蛋白酶裂解位点位于膜的细胞质侧，距离17000道尔顿肽段的N端2000道尔顿。这一信息使得能够在带3的一级结构中定位15000道尔顿片段的多个确定位点。这些位点支持了该肽段在双层膜中折叠的观点。
