ABSTRACT: In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1-10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h. The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant. In the presence of 25-50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level. If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate. After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease. In the presence of coformycin, erythrocytes utilized adenosine at a rate of 0.2-0.3 mmol/l cells h. Their adenylate pool increased at a rate of 0.10-0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly. The energy charge of cells decreased significantly from 0.86 ± 0.05 (control) to 0.82 ± 0.06. Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02-5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02-0.35 mmol/l cells h. The energy charge of these erythrocytes increased significantly up to 0.91 ± 0.03. After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2-3 h.
   © 1999 Academic Press

Keywords: Adenosine, ATP, adenylate pool, energy charge, human erythrocytes.

Reprint requests to: Dr. Victor M. Vitvitsky, Research Center for Hematology of RAMS, Moscow, 125167 Russia, fax: (7-095) 212-88-70, e-mail: victor@blood.ru.

ABSTRACT: The effects of bacterial cells, extracellular products (ECP) and a purified cysteine protease of Vibrio harveyi on hemostasis and plasma components of tiger prawn (Penaeus monodon) were studied. The clotting ability of the hemolymph withdrawn from moribund prawns pre-injected with the bacteria, ECP, cysteine protease or PBS (control) was observed for 2 h at 25 C. Of these, only the control group was clottable while all the other groups were unclottable. A component of the plasma, previously identified as coagulogen-like protein, was further confirmed to be a coagulogen by the comparison of plasma with serum on non-reduced SDS-PAGE or using rabbit antiserum to the coagulogen-like protein (R alpha coagulogen) to neutralize the clotting ability of normal prawn hemolymph. The coagulogen was reduced in amount in plasma of moribund prawns after injection with the bacteria, ECP or cysteine protease while it apparently disappeared after pre-incubation with the ECP or cysteine protease for 2 h at 25 C compared with normal prawn plasma as observed in crossed immunoelectrophoresis (CIE) gels. The reduction of the amount of coagulogen in plasma of moribund prawns was also evident in CIE gels using R alpha; coagulogen. In addition, the apparent disappearance of the coagulogen mentioned above was eventually proven to be due to the change of its migration rate in CIE gels after pre-incubation with ECP or cysteine protease, since the disappeared coagulogen arc (arc 2) (migrated into arc 1) could be visualized by using R alpha coagulogen or by reducing the time for pre-incubation from 2 h to 30 min. Thus, the effects of cysteine protease on plasma coagulogen observed in vitro and in vivo may markedly interfere with hemostasis leading to the occurrence of unclottable hemolymph. These complex events may significantly contribute to the pathogenicity of V. harveyi in the prawn.
   © 1999 Academic Press

Keywords: Coagulogen, cysteine protease, extracellular products, hemostasis, Penaeus monodon, Vibrio harveyi.

Reprint requests to: Kuo-Kau Lee, Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung, TAIWAN, fax: 008862 24633150, e-mail: kklee@ntou66.ntou.edu.tw.