SYNTHESIS, ISOLATION, AND CHARACTERIZATION OF ENDOGENOUS BETA-GALACTOSIDE-BINDING LECTINS IN HUMAN-LEUKOCYTES

被引:21
作者
ALLEN, HJ
GOTTSTINE, S
SHARMA, A
DICIOCCIO, RA
SWANK, RT
LI, H
机构
[1] ROSWELL PK CANC INST, DEPT GYNECOL ONCOL, BUFFALO, NY 14263 USA
[2] ROSWELL PK CANC INST, DEPT CELL & MOLEC BIOL, BUFFALO, NY 14263 USA
[3] CANISIUS COLL, DEPT BIOL, BUFFALO, NY 14208 USA
关键词
D O I
10.1021/bi00100a026
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Galaptin, a beta-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5K), pI (4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonal antiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric beta-galactoside-binding protein of M(r) 15.5-16.5K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chromatography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by the polymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived amino acids corresponded to residues 6-65, 84-114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface. The results presented here demonstrate that galaptin is present in a variety of leukocytes including buffy coat cells. Although buffy coat cells may accumulate galaptin, they do not synthesize it in vitro. The 15.5-16.5-kDa beta-galactoside-binding lectin that is synthesized does not appear to accumulate in the buffy coat cells, and it may be a secretory protein [Allen, H. J., et al. (1986) Immunol. Invest. 15, 123-138].
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页码:8904 / 8910
页数:7
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