Vibrio hollisae produces a hemolysin (Vh-rTDH) that is related to the thermostable direct hemolysin of Vibrio parahaemolyticus (Vp-TDH). Although both hemolysins are essentially similar biologically and immunologically, they differ markedly in heat stability; Vp-TDH is heat stable, whereas Vh-rTDH is heat labile. To elucidate the relationships between their characteristics and molecular structures, we analyzed the amino acid sequence of Vh-rTDH and compared it with that of Vp-TDH. Vh-rTDH consisted of 165 residues, of which 23 residues, spread over the peptide chain, differed from those of Vp-TDH.A halophilic vibrio, Vibrio hollisae, has been isolated from stool cultures in cases in which no other enteric pathogens were identified (1). In most cases, patients were known to have eaten raw seafood a few days before they became ill, and the clinical characteristics of the disease resembled those in cases of gastroenteritis caused by non-01 Vibrio cholerae (3,5,6).In searching out pathogenic factors, we found that non-01 V. cholerae and V. hollisae produce Vp-TDH (Vibrio parahaemolyticus thermostable direct hemolysin)-related toxins . Since Vp-TDH has been considered the most probable pathogenic factor in Vibrio parahaemolyticus infection, then by analogy, both NAG-rTDH and Vh-rTDH are also thought to be pathogenic factors. We purified these Vp-TDH-related hemolysins and characterized them (13,14). These three hemolysins all have the same molecular mass (approximately 40,000 in dimer form) and are immunologically similar but differ physicochemically. The most interesting difference is the thermal stability in these hemolysins. Vh-rTDH is heat labile, whereas both Vp-TDH and NAG-rTDH are heat stable: Vp-TDH retains 91% hemolytic activity after incubation at 70°C for 10 min, whereas the activity is completely lost with 14).The complete amino acid sequence of Vp-TDH has been determined (11) and deduced by nucleotide sequence analysis of its structural gene (7). We therefore undertook an analysis of the amino acid sequence of Vh-rTDH to clarify the differences in the primary structure between this heatlabile hemolysin and the heat-stable Vp-TDH. Here we report the result of the amino acid sequence analysis of Vh-rTDH and comparison of its sequence with that of Vp-TDH.Vh-rTDH and Vp-TDH were purified from the culture supernatants of V. hollisae RIMD 2221001 and V. parahaemolyticus RIMD 2210086, respectively, as described previously (2,14). Achromobacter protease I (lysylendopeptidase) and Staphylococcus aureus V8 protease were obtained from Wako Pure Chemicals, and Miles Laboratories, Inc., respectively. Columns used for high-performance liquid * Corresponding author. chromatography were ,u-Bondasphere C4-100 (3.9 by 150 mm, 15 ,um; Nihon Waters Ltd.) and Bakerbond wide-pore butyl (4.6 by 250 mm, 30 nm; Baker Chemical Co.).Intact hemolysin (10 nmol) was digested with Achromobacter protease I for 6 h at 30°C in 0.2 ml of 0.01 M Tris hydrochloride buffer (pH 9.0), at an enzyme-to-substrate molar ratio of 1:400. Digestion w...