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At least 433 records · Page 24Linked to original sources
PubMed1990

The pancreatic membrane protein GP-2 localizes specifically to secretory granules and is shed into the pancreatic juice as a protein aggregate.

GP-2 is the major secretory granule membrane glycoprotein of the exocrine pancreas and appears in the pancreatic juice in a modified sedimentable form. We have localized GP-2 in the rat pancreas at the electron microscopic level using affinity-purified antibodies and found it to be concentrated in the zymogen granules and in the acinar lumen. Label was also present on the apical and basolateral plasma membranes but prior treatment of the sections with periodate to eliminate the contribution of highly antigenic oligosaccharide moieties reduced substantially the staining of the basolateral surface. Approximately 45% of the GP-2 in the granules was not membrane-associated but appeared instead in the granule lumen. Parallel biochemical characterization of GP-2 in isolated secretory granules demonstrated that 60% fractionated with the membranes after granule lysis while 40% remained in the content fraction. Unlike the membrane-associated form of the protein, which is linked to the membrane via glycosyl-phosphatidylinositol (GPI), GP-2 in the content did not enter the detergent phase upon Triton X-114 extraction; nor was it sedimentable at 200,000g, as is characteristic of the form collected in the pancreatic juice. In addition, GP-2 in the pancreatic juice was recovered in the aqueous phase during Triton X-114 extraction and yet remained sedimentable after detergent extraction, demonstrating that its ability to remain in large aggregates was independent of lipid. These results are consistent with a life cycle for the protein that begins with synthesis of a membrane-associated precursor that can be converted by lipolytic or proteolytic cleavage to a soluble form within the zymogen granule. Further modification to a sedimentable form may then occur in the pancreatic juice.

Animals↗
PubMed1997

Synthesis and characterization of supramolecular protein aggregates: self-assembled, molecularly-ordered, tubes from electrostatic complementation of glutamine synthetase dodecamers.

Dodecameric Escherichia coli glutamine synthetase (GS) is formed from identical subunits arranged in face-to-face hexameric rings. In the presence of Zn2+ and other transition metal ions the individual dodecamers 'stack' to form protein tubes. Previous results have suggested that six binuclear intermolecular metal binding sites are generated at each dodecamer-dodecamer interface by juxtaposition of the N-terminal helices of each subunit adjacent to an analogous helix from a docked dodecamer. In principle, replacement of one of the metal binding sites within each pair of helices with charged amino acids could generate electrostatic interactions that would provide the basis for heterospecific protein-protein interactions. In turn, this would allow for ordered assembly of protein tubes with alternating, chemically distinguishable, components. This hypothesis was tested by replacement of one of the metalligating histidines (His12) with aspartic acid, arginine or cysteine. The H12C mutant was further elaborated by selective thiol modification, with either of the charged reagents 2-iodo-acetic acid or 2-chloro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12. Light scattering and electron microscopy were used to monitor the 'stacking ability' of these variants in the presence of Zn2+. No, or few, GS 'tubes' were observed in solutions containing only H12D, H12R, H12C-CA or H12C-IA, in the presence or absence of Zn2+. In contrast, in mixtures containing H12C-CA and either H12D or H12C-IA, the complementary GS variants stack in the presence of 100 microM Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers. Fluorescence energy transfer experiments with fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA (acceptor) were performed and compared with the energy transfer efficiency with mixtures containing variable ratios of acceptor-labeled and donor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangements of donor and acceptor. The efficiency of metal-dependent energy transfer in mixtures containing the acceptor-labeled H12C-CA and the donor-labeled H12C-IA was 3.2-fold greater than expected for a random distribution of charged variants. Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction between chemically distinguishable dodecamers that align in an ordered one-dimensional array.

Binding Sites↗
PubMed1990

Drug-protein interactions. Comparative studies of levamisole and structural analogs or agents with immunomodulatory effect on the mechanism of protein aggregation.

Interactions of tetramisole, imidazo[2,1-b]thiazolic derived or immunomodulatory agents with human serum albumin (HSA) and immunoglobulins G and A has been investigated in vitro using polyacrylamide gel electrophoresis and immunoelectrophoresis. The results suggest that only the chemical structure type of tetramisole is able to induce a protein-protein interaction described as a mechanism of disulfide-sulfhydryl interchange reactions. The groups of atoms involved in the interaction are characterized.

Blood Proteins↗
PubMed1997

The relationship between protein aggregation and molecular mobility below the glass transition temperature of lyophilized formulations containing a monoclonal antibody.

PURPOSE: To find out if the physical instability of a lyophilized dosage form is related to molecular mobility below the glass transition temperature. Further, to explore if the stability data generated at temperatures below the glass transition temperature can be used to predict the stability of a lyophilized solid under recommended storage conditions. METHODS: The temperature dependence of relaxation time constant, tau, was obtained for sucrose and trehalose formulations of the monoclonal antibody (5 mg protein/vial) from enthalpy relaxation studies using differential scanning calorimetry. The non-exponentiality parameter, beta, in the relaxation behavior was also obtained using dielectric relaxation spectroscopy. RESULTS: For both sucrose and trehalose formulations, the variation in tau with temperature could be fitted Vogel-Tammann-Fulcher (VTF) equation. The two formulations exhibited difference sensitivities to temperature. Sucrose formulation was more fragile and exhibited a stronger non-Arrhenius behavior compared to trehalose formulation below glass transition. Both formulations exhibited < 2% aggregation at t/tau values < 10, where t is the time of storage. CONCLUSIONS: Since the relaxation times for sucrose and trehalose formulations at 5 degrees C are on the order of 10(8) and 10(6) hrs, it is likely that both formulations would undergo very little (< 2%) aggregation in a practical time scale under refrigerated conditions.

Antibodies, Monoclonal↗
PubMed2000

Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.

The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that deltaN6beta2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of beta2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between beta-strands far removed from this constrain is greatly perturbed.

Amino Acid Sequence↗