Mature amyloid fibrils are believed to be formed by the lateral association of discrete structural units designated as protofibrils, but this lateral association of protofibrils has never been directly observed. We have recently characterized a thioesterase from Alcaligenes faecalis, which was shown to exist as homomeric oligomers with an average diameter of 21.6 nm consisting of 22 kDa subunits in predominantly beta-sheet structure. In this study, we have shown that upon incubation in a 75% ethanol solution, the oligomeric particles of protein were transformed into amyloid-like fibrils. TEM pictures obtained at various stages during fibril growth helped us to understand to a certain extent the early events in the fibrillization process. When incubated in 75% ethanol, oligomeric particles of protein grew to approximately 35-40 nm in diameter before fusion. Fusion of two oligomers of 35-40 nm resulted in the formation of a fibril. Fibril formation was accompanied by a reduction in the diameter of the particle to approximately 20-25 nm along with concomitant elongation to approximately 110 nm, indicating reorganization and strengthening of the structure. The elongation process continued by sequential addition of oligomeric units to give fibers 500-1000 nm in length with a further reduction in diameter to 17-20 nm. Further elongation resulted in the formation of fibers that were more than 4000 nm in length; the diameter, however, remained constant at 17-20 nm. These data clearly show that the mature fibrils have assembled via longitudinal growth of oligomers and not via lateral association of protofibrils.

Long-chain acyl-CoA thioesterases (EC 3.1.2.2) hydrolyze acyl-CoA esters to nonesterified fatty acids and coenzyme A (CoASH) [1]. These are ubiquitously expressed in bacteria, yeast, plants and mammals, and in most cell compartments, such as endoplasmic reticulum, cytosol, mitochondria and peroxisomes. Several unrelated thioesterases have been purified to homogeneity from plants, animals, and bacteria, and the cDNAs encoding several of them have been cloned and sequenced [2][3][4][5][6][7]. Although the physiological functions of these enzymes remain largely unknown, it is speculated that they regulate lipid metabolism by maintaining appropriate concentrations of acyl-CoA, CoASH, and nonesterified fatty acids. The only established function for acyl-CoA thioesterases is in the termination of fatty acid synthesis in eukaryotes [8].Two thioesterases, I and II, that cleave acyl-CoA molecules in vitro have been characterized from A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS ⁄ MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C 12 to C 18 chain length with V max and K m of 3.58-9.73 lmolAEmin )1 AE(mg protein) )1 and 2.66-4.11 lm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65°C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70°C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C 2 to C 12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20°C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.Abbreviations DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); TEM, transmission electron microscopy.