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Titolo:
IDENTIFICATION OF DOMAINS IN HUMAN BETA-HEXOSAMINIDASE THAT DETERMINESUBSTRATE-SPECIFICITY
Autore:
PENNYBACKER M; LIESSEM B; MOCZALL H; TIFFT CJ; SANDHOFF K; PROIA RL;
Indirizzi:
NIDDK,SECT BIOCHEM GENET,GENET & BIOCHEM BRANCH,NIH,BLDG 10,RM 9D-20,10 CTR DR MSC 1810 BETHESDA MD 20892 NIDDK,SECT BIOCHEM GENET,GENET & BIOCHEM BRANCH,NIH BETHESDA MD 20892 UNIV BONN,INST ORGAN CHEM & BIOCHEM D-53121 BONN GERMANY
Titolo Testata:
The Journal of biological chemistry
fascicolo: 29, volume: 271, anno: 1996,
pagine: 17377 - 17382
SICI:
0021-9258(1996)271:29<17377:IODIHB>2.0.ZU;2-U
Fonte:
ISI
Lingua:
ENG
Soggetto:
TAY-SACHS DISEASE; ACTIVATOR PROTEIN; EXTENSIVE HOMOLOGY; EXPRESSION; DEGRADATION; GENES; GM2;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
20
Recensione:
Indirizzi per estratti:
Citazione:
M. Pennybacker et al., "IDENTIFICATION OF DOMAINS IN HUMAN BETA-HEXOSAMINIDASE THAT DETERMINESUBSTRATE-SPECIFICITY", The Journal of biological chemistry, 271(29), 1996, pp. 17377-17382

Abstract

The lysosomal beta-hexosaminidases are dimers composed of alpha and beta subunits, beta-Hexosaminidase A (alpha beta) is a heterodimer, whereas hexosaminidase B (beta beta) and S (alpha alpha) are homodimers, Although containing a high degree of amino acid identity, each subunitexpresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the beta-subunit primarily degrades neutral substrates, whereas the alpha-subunitsite is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates, Among human isozymes, only beta-hexosaminidase A together with the G(M2) activator protein can degrade the natural substrate, G(M2) ganglioside, at physiologically significant rates. To identify the domains of the human beta-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both alpha- and beta-subunit sequences, The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade G(M2) ganglioside in the presence of G(M2) activator protein, Generation of activity against the sulfated substrate required the substitution of two noncontinuous alpha-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the beta-subunit, Chimeric constructs containing only one of these regions linked to the beta-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529), Neither the chimeras nor the wild-type subunits displayed activator-dependent G(M2)-hydrolyzing activity when expressed alone, However, one chimeric subunit containing alpha amino acids 1-191 fused with beta amino acids 225 to 556, when co-expressed with the wild-type alpha-subunit, showed activity comparable with that of recombinant beta-hexosaminidase A formed by the co-expression of the alpha- and beta subunits. This result indicates that the beta-subunit amino acids 225-556 contribute an essential function in the G(M2)-hydrolyzing activity of beta-hexosaminidase A.

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Documento generato il 01/12/20 alle ore 19:53:33