ABSTRACT
The product of the human ob (obesity) gene, leptin, appears to function in the maintenance of body weight in vivo. When injected into mice, this hormone reduces food consumption and causes weight loss. This work has been done with recombinant leptin (re-leptin) purified and renatured from inclusion bodies in Escherichia coli. We have expressed the human obesity gene encoding the predicted full-length leptin in Spodoptera frugiperda (Sf-9) cells by infection with the recombinant baculovirus system. Protein corresponding to re-leptin was secreted into the culture medium and purified in sufficient quantity for testing biological activity. The secreted re-protein was characterized and found to be unmodified except for correct cleavage of the signal peptide during export from the cells. The resulting molecule is expected to be properly folded and has been purified to a high level of homogeneity. The re-leptin secreted from Sf-9 cells should be an appropriate source of protein for study of the native structure.
Subject(s)
Baculoviridae/genetics , Proteins/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Circular Dichroism , Cloning, Molecular , Humans , Leptin , Molecular Sequence Data , Oligodeoxyribonucleotides , Peptide Mapping , Proteins/genetics , Proteins/metabolism , Spectrophotometry, Ultraviolet , Spodoptera , Trypsin/chemistryABSTRACT
The equilibrium denaturation of human insulin in a monomer-inducing solvent and of two monomeric insulin analogs, lysB28proB29 insulin and aspB10desB28-30 insulin, was reexamined [Brems, D. N., Brown, P. L., Heckenlaible, L. A., & Frank, B. H. (1990) Biochemistry 29,9289-9293] by circular dichroism (CD) at additional wavelengths in the near-UV region. Previous denaturation studies were limited by the solubility of guanidine hydrochloride being only slightly greater than the level of denaturant required to fully unfold human insulin; therefore, only a few data points were available for construction of the post-transitional baseline. In the present study, we report the use of an unfolded mimic created by enzymatic digestion of insulin to confirm the slope of the post-transitional baseline. Evidence for equilibrium unfolding intermediates for each of these insulins was indicated by noncoincidence of the denaturation transitions as monitored by tyrosine and helical-dependent CD bands (270 and 224 nm, respectively). Additional evidence for intermediates through multiphasic denaturation transitions was obtained at a wavelength likely related to disulfide conformation, 251 nm. The results suggest that for each of the insulins, at least two intermediates are significantly populated. An unfolding model is proposed in which the conformation of the least stable intermediate is slightly unfolded only in the C-terminal segment of the B chain. A second more stable intermediate retains minimal secondary structure while containing localized structure proximal to one or more of the disulfide groups. The presence of equilibrium intermediates has important implications for the folding pathway of insulin, in pharmaceutical applications such as formulation stability, and for conformational transitions that accompany receptor binding.
Subject(s)
Insulin/chemistry , Animals , Circular Dichroism , Guanidine , Guanidines , Humans , Insulin/analogs & derivatives , Male , Protein Denaturation , Protein Folding , Rats , Rats, Sprague-Dawley , Spectrophotometry, UltravioletABSTRACT
Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.
Subject(s)
Insulin/chemistry , Protein Structure, Tertiary , Amino Acid Sequence , Cystine , Drug Stability , Insulin/analogs & derivatives , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Polymers , Protein Conformation , Protein Denaturation , Temperature , ThermodynamicsABSTRACT
The collective effectiveness of several heat stress prevention measures has been evaluated. The relative merits of both physical and administrative controls and their interrelationships have been explored. It is concluded that a combination of physical and administrative control is more effective than any single physical or administrative control in managing heat stress. The use of the preventive measures discussed provided protection to a labor force of 120 employees working approximately 2.4 million man-hours over a six-year period where temperatures average 26.6 degrees C Wet Bulb Globe Temperature (WBGT) during the nonsummer months and 34.4 degrees C WBGT during the summer months. Only three documented cases of heat stress were encountered during this period.
