Identification of the cDNA for human red blood cell-specific hexokinase isozyme.
A unique cDNA for hexokinase (HK) was identified from poly(A)+ RNA of human reticulocytes by anchored polymerase chain reaction. This appeared to represent the cDNA for the red blood cell (RBC)-specific HK isozyme (HKR) described in our previous study (Murakami et al: Blood 75:770, 1990). Its nucleotide sequence was identical to HKI cDNA except for the 5' extreme end. It lacked the first 62 nucleotides of the HKI coding region: instead, it contained a unique sequence of 60 nucleotides at the beginning of the coding sequence as well as another unique sequence upstream of the putative translation initiation site. It lacked the porin-binding domain which facilitates binding to the mitochondria, thus explaining the exclusive cytoplasmic localization of HKR. It was the major cDNA derived from reticulocytes, consistent with the observation that HKR activity is predominant in reticulocytes. Northern blot analysis showed that it was expressed in the reticulocytes and in the K562 erythroleukemic cell line, but not in a lymphocytic cell line. In the extract of K562 cells, HKR activity co-eluted with the HKR of human RBCs on a MonoQ column (Pharmacia, Piscataway, NJ) chromatography, using a salt gradient elution. The separate genetic control of the RBC-specific HK isozyme explains the clinical reports of two types of HK deficiency, one in which the HK activity was reduced exclusively in the RBC (HKR defect) and another with general decrease of HK activity in several tissues (HKI defect).
Murakami
K
K
Division of Pediatric Hematology, College of Physicians & Surgeons of Columbia University, New York 10032, USA.
Piomelli
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000097:blood / Q000302:isolation & purification*; D004912:Erythrocytes / Q000201:enzymology* / Q000378:metabolism; D006593:Hexokinase / Q000097:blood* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007527:Isoenzymes / Q000097:blood* / Q000235:genetics* / Q000302:isolation & purification; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis; D012156:Reticulocytes / Q000201:enzymology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D006593:Hexokinase