Abstract
The glutamate dehydrogenase from a single human liver has been studied. The subunit size was found to be 55,200 +/- 1,500 by sedimentation equilibrium. The partial specific volume is 0.732 as calculated from the amino acid composition. The sequence was determined by isolation of peptides after cyanogen bromide (CNBr) cleavage; the fraction containing the largest peptides was hydrolyzed by trypsin after maleylation. Studies on these peptides accounted for 454 residues of the 505 residues that are presumably present in the protein. For the 51 residues that were not represented in isolated peptides, we have tentatively assumed that the sequence is the same as that of the bovine enzyme. Methionine and arginine residues in these peptides could be placed on the basis of the specificity of cleavage by CNBr or trypsin. In all, 349 residues were placed in sequence, and were aligned by homology with the corresponding peptides of the bovine and chicken enzymes. From the present information, there are 24 known differences in sequence between the human and bovine enzymes and 41 between the human and chicken enzymes. In addition, the human enzyme contains 4 additional residues at the NH2 terminus as compared to the bovine enzyme. In a peptide from the human enzyme, an additional residue, isoleucine 385, was detected by automated Edman degradation. Reinvestigation of the bovine sequence demonstrated that this residue is also present in the bovine enzyme (and presumably in the chicken enzyme also). Residue 384 of the bovine enzyme, previously reported as Glx has now been shown to be glutamine.
Highlights
From the Department of Biological Chemistry, UCLA School of Medicine, and the Molecular Biology Institute, University of California, Los Angeles, California 90024
The sequence was determined by isolation of peptides after cyanogen bromide (CNBr) cleavage; the fraction containing the largest peptides was hydrolyzed by trypsin after maleylation
In this study of the glutamate dehydrogenase from a single human liver, it was possible to isolate from the mixture of cleavage products after cyanogen bromide treatment peptides representing 162 residues of the protein
Summary
Residues -4 through 12 (Peptides CN 1, CN lA, and CN 1B)-Peptide CN 1B is from the cleavage of the Asp-Pro bond between residues 6 and 7 (Fig. 1A). The sequence was determined by carboxypeptidase action and by eight steps of Edman degradation on Peptide CN 1. Residues 13 through 19 (Peptide TM 2B)-Two manual Edman steps and hydrolysis with carboxypeptidases A + B yielded the sequence. Residues 36 through 42 (Peptide TM 5)-No residue was released by Edman degradation because of cyclization of NH2terminal glutamine by analogy with residue 36 of bovine glutamate dehydrogenase. The first 8 residues of Peptide TM 8A were identified by Edman degradation (Table VIII). From these data and the results with carboxypeptidase Y (Table IX), most of the sequence was determined. Residues 68 through 79 (Peptide TM 9)-One step of Edman degradation yielded aspartic acid; no additional residue was released.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
