Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase.
I have isolated glucose-6-phosphate dehydrogenase from rabbit liver microsomes and determined its complete amino acid sequence. Sequence determination was achieved by automated Edman degradation of peptides generated by chemical and enzymatic cleavages. The microsomal enzyme consists of 763 residues and is quite dissimilar from the previously characterized cytosolic enzymes. The N terminus of the microsomal enzyme is blocked by a pyroglutamyl residue. Carbohydrate is attached at Asn-138 and Asn-263, implying that the bulk of the protein is oriented on the lumenal side of the endoplasmic membrane. The amino acid sequence of the microsomal protein shows limited homology to the extensively sequenced cytosolic glucose-6-phosphate dehydrogenases. Clusters of up to six identical residues can be identified in four regions: peptide segments at residues 10-21, 154-163, and 173-261. In addition, another array of identical residues, requiring a 100-residue deletion in the sequence of the microsomal enzyme, spans residues 436-462 and corresponds to residues 348-373 of the cytosolic protein. Two segments with a Gly-Xaa-Gly-Xaa-Xaa-Gly motif, related to a coenzyme binding fold, were identified at Gly-399 and Gly-491. In the cytosolic enzymes, a variation of this sequence motif occurs at Gly-37 and Gly-241. The 300-residue C-terminal segment of the microsomal enzyme is unique and has no counterpart in the cytosolic or the bacterial enzymes. An unexpected finding with regard to the microsomal enzyme is that it lacks an identifiable membrane-spanning region or the lumenal-protein C-terminal consensus sequences Lys-Asp-Glu or His-Ile/Thr-Glu-Leu. Thus, the mode of transport and retention of this protein in the lumen of endoplasmic reticulum remains to be determined.
Ozols
J
J
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030-3305.
D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D005954:Glucosephosphate Dehydrogenase; D010450:Endopeptidases