United States Patent ( 10 ) Patent No . : US 7479 , 375 B 2
(54) METHOD FOR CLASSIFYINGA 5,372,946 A 12/1994 Cusak et al. MCROORGANISM IN A BIOLOGICAL 5,389,547 A 2f1995 Tanaka et al. SAMPLE 5,401,647 A 3, 1995 Tanaka et al. 5,474,984 A 12/1995 Tanaka et al. (75) Inventors: Norman R. Wainwright, Falmouth, MA 5,504,011 A 4, 1996 Gavin et al. 5,518,006 A 5/1996 Mawhirt et al. (US); Dana M. Nutter, Charleston, SC 5.534.226 A 7/1996 Gavin etal (US) 5,550,030 A 8/1996 Tanaka et al. 5,574,023 A 11/1996 Shibata et al. (73) Assignee: Charles River Laboratories, Inc., 5,591.403 A 1/1997 Gavin et al. Wilmington, MA (US) 5,591,628 A 1/1997 Baek et al. 5,605,806 A 2/1997 Tanaka et al. (*) Notice: Subject to any disclaimer, the term of this 5,637,474 A 6/1997 Takaoka et al. patent is extended or adjusted under 35 D380,555 S 7/1997 Kurosaki et al. U.S.C. 154(b) by 264 days. 5,681,710 A 10/1997 Tanaka et al. 5,695,948 A 12/1997 Tanaka et al. (21) Appl. No.: 11/331,600 5,702,882 A 12/1997 Tamura et al. D390,661 S 2/1998 Foggia (22) Filed: Jan. 13, 2006 (Continued)
In the spiny lobster (Panulirus interruptus) the prophenoloxidase is located in plasma not in haemocytes.
In the spiny lobster (Panulirus interruptus), unlike other crustaceans most of the prophenoloxidase (proPO) was detected in cell-free plasma (86.3%). In spite of its location, lobster proPO activating system has a similar activation mechanism to other crustacean proPO systems. Haemocyte lysate was able to activate the plasma proPO indicating location of the prophenoloxidase activating enzyme (PPAE) in haemocytes. Lobster haemocyte PPAE was isolated by affinity chromatography and its participation as activating enzyme was demonstrated. This enzyme is a serine-proteinase that transforms the inactive form (proPO) to an active one (phenoloxidase). The PPAE was also present in the cell-free supernatant of haemocytes previously incubated with Vibrio alginolyticus.
The prophenoloxidase activating system in crayfish
Abstract 1. 1. A preparation (designated 0–40 fraction) containing stable prophenoloxidase (proPO) and other dormant components of the prophenoloxidase activating system was obtained from crayfish hemocytes. 2. 2. Activation of proPO in the 0–40 fraction was elicited by β1,3-glucans, SDS, trypsin or heat, while a protease inhibitor, p-NPGB, inhibited activation of proPO by β1,3-glucans but, not activation by SDS or heat. 3. 3. Ca2+ was always necessary for the activation of proPO and treatment of the 0–40 fraction with EDTA caused irreversible inactivation of proPO activating system, seemingly leaving proPO intact. 4. 4. The enzyme responsible for activating proPO could be separated from proPO and this enzyme was found to be inhibited by p-NPGB. This enzyme could activate proPO in the 0–40 fraction treated with EDTA. 5. 5. Protease activity increased more than 10-fold in the 0–40 fraction after the incubation with β1,3-glucans and Ca2+. 6. 6. The proPO activating system may operate as a recognition system in crayfish, and it is suggested that this system functions as a complement-like system in arthropods.