Preparation of recombinant rat eosinophil-associated ribonuclease-1 and -2 and analysis of their biological activities.
Rat eosinophils contain eosinophil-associated ribonucleases (Ears) in their granules. Ears are thought to be synthesized as pre-forms and stored in the granules as mature forms. However, the N-terminal amino acid of mature Ear-1 and Ear-2 is still controversial. Therefore, we prepared two recombinant mature forms of Ear-1 and Ear-2 in which the N-terminal amino acids are Ser24 (S) [Ear-1 (S) and Ear-2 (S)] and Gln26 (Q) [Ear-1 (Q) and Ear-2 (Q)], and analyzed their biological activities by comparing them with those of pre-form Ear-1 and pre-form Ear-2. The four mature Ears showed RNase A activity as well as bovine pancreatic RNase A activity, but pre-Ear-1 and pre-Ear-2 showed no RNase A activity. Mature Ear-1 (Q) and mature Ear-2 (Q) showed more potent RNase A activity than mature Ear-1 (S) and mature Ear-2 (S), respectively. The RNase A activities of mature Ear-1 (Q) and mature Ear-2 (Q) were reduced by treatment at 96 degrees C for 20 min or with RNase inhibitor. The growth of Escherichia coli was inhibited by both pre-Ears and mature Ears in a concentration-dependent manner, and was almost completely suppressed at 1.0 microM. The bactericidal activities of mature Ear-1 (Q) and mature Ear-2 (Q) were not inhibited by RNase inhibitor, but was increased by treatment at 96 degrees C for 20 min.
Ishihara
Kenji
K
Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Asai
Kanako
K
Nakajima
Masahiro
M
Mue
Suetsugu
S
Ohuchi
Kazuo
K
D000595:Amino Acid Sequence; D000818:Animals; D001770:Blood Bactericidal Activity; D001798:Blood Proteins; D002417:Cattle; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000201:enzymology / Q000378:metabolism*; D004926:Escherichia coli; D005998:Glycine / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D012358:RNA, Transfer, Met / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000097:blood / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases; D012441:Saccharomyces cerevisiae; D012694:Serine / Q000378:metabolism
D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D012358:RNA, Transfer, Met; D011994:Recombinant Proteins; D012694:Serine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D005998:Glycine