ABSTRACT
The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.
Subject(s)
Actins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Actins/antagonists & inhibitors , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Carrier Proteins/genetics , Centrifugation , Dictyostelium , Iodine Radioisotopes , Microfilament Proteins/genetics , Molecular Sequence Data , Molecular Weight , Peptide Mapping , Polymers/chemistry , Polymers/metabolism , Protein Binding , Pyrenes/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemical synthesis , Recombinant Proteins/metabolism , Sequence Deletion , Solutions , ViscosityABSTRACT
Metastatic malignant pheochromocytoma can be a disease of long duration and though poorly responsive to both radiation therapy and chemotherapy, pharmacologic management of its myriad manifestations can be very effective. Hypertension is the most recognized feature of this disease, but gastrointestinal manifestations can, on rare occasions, be just as serious and life threatening. We present two patients with metastatic pheochromocytoma who developed pseudo-obstruction, and discuss the pathopharmacology and treatment of this syndrome.
