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Osmotic effects on arginine kinase function in living muscle of the blue crab Callinectes sapidus

Stewart M. Holt^* and Stephen T. Kinsey

Department of Biological Sciences, University of North Carolina at Wilmington, 601 South College Road, Wilmington, NC 28403-3297, USA
^* Present address: Department of Biology, University of Vermont, Burlington, VT 05405, USA

View larger version (14K): [in a new window]   Fig. 1. Representative 31P-NMR spectra arranged in time series during an extreme hypo-osmotic treatment. From left to right, the peaks are sugar phosphate, inorganic phosphate (Pi), arginine phosphate (tallest peak) and the , and ß peaks of ATP. The tissue was acclimated to 35 salinity and exposed to a medium simulating a transfer to 5. Each spectrum was collected over a period of 10 min, so the total experimental time was 2 h. The first spectrum was collected under control conditions in which the tissue was superfused with a medium of 960 mosmol l-1, and all subsequent spectra were collected while the muscle was superfused with a medium of 640 mosmol l-1. Several such stability experiments were conducted for the hypo- and hyperosmotic treatments and, in each case, the arginine phosphate, ATP and Pi peak areas remained constant. This indicates that the tissues were not energetically compromised during the flux experiments.

View larger version (9K): [in a new window]   Fig. 2. Typical spectra used for the measurement of the pseudo-first-order unidirectional rate constant for the forward direction (Kforward) of the arginine kinase reaction. The spectrum on the left is a control in which the saturating irradiation is applied at a frequency offset from the arginine phosphate resonance equivalent to that between the arginine phosphate and -ATP resonances (vertical arrow). The spectrum on the right demonstrates that, when the -P of ATP is saturated with radiation, a decrease in the arginine phosphate peak is induced that is proportional to the rate of transfer of the phosphate from arginine phosphate to ADP (forward arginine kinase reaction). To measure Kreverse, the arginine phosphate peak is saturated.

View larger version (26K): [in a new window]   Fig. 3. Linear regression analysis of arginine kinase rate constants and flux rates against the extent of the hypo- or hyperosmotic treatment (osmotic change). The osmotic change was determined by subtracting the control osmolarity from the experimental osmolarity. Negative values indicate a hypo-osmotic treatment, positive values indicate a hyperosmotic treatment and zero indicates controls. For example, the-320mosmoll-1 osmotic treatment results when muscle from an animal exposed to 35 salinity is superfused with a medium of 640mosmoll-1 (640-960=-320). Regression lines are shown with 95% confidence limits. Regression equations are as follows: kforward=-4.4x10-5O+0.10, r2=0.07, P=0.07 (not significant); kreverse=-3.3x10-4O+0.41, r2=0.28, P<0.01; forward flux=-1.3x10-3O+1.43, r2=0.24, P<0.01; reverse flux=-1.4x10-3O+1.66, r2=0.22, P<0.01, where O is osmotic change.

View larger version (9K): [in a new window]   Fig. 4. Example of a 1H-NMR spectrum of a living muscle from a blue crab that had been acclimated to a salinity of 35. The large peak at 3.2p.p.m. varied in amplitude with salinity and has been tentatively assigned to betaine. Note the small peak at 3.85p.p.m., which is also indicative of betaine.

View larger version (9K): [in a new window]   Fig. 5. The effect of acclimation salinity and experimental osmolarity on the relative concentration of betaine in crab muscle. +, muscles exposed to 960mosmoll-1; x, muscles exposed to 720mosmoll-1; , muscles exposed to 640mosmoll-1. The horizontal bars are mean values for each acclimation salinity. The units on the y-axis are arbitrary. Two-way ANOVA indicated a significant effect of acclimation salinity but no effect of experimental osmolarity. N=3 for each treatment.