ISOLATION OF SALT GLANDS AND GUT TUBES FROM BRINE SHRIMP (ARTEMIA SALINA)

May S. ARNAUT

The primary objective of this work is to isolate at different times of development the ion transporting tissues in the brine shrimp that are abundant in Na, K­ATPase. This is important since it is demonstrated that purification of the enzyme to homogeneity requires such an isolation. In the kidney, dog, sheep or guinea pig, the specific activity in the starting material is cri­tical for the success of the purification of the enzyme to homogeneity. Isolation of the enzyme rich kidney medulla with a specific activity of 1-5 uM Pi/mg protein/ hour is an essential first step. This usually achieves a specific activity of 15-40 umoles Pi/mg pro­tein/hour in the crude membrane fraction and a specific activity of ~ 2000 umoles Pi/mg protein/hour in the pure enzyme. Attempts at purifying the enzyme from whole kidney and whole nauplii have failed in achieving the desired purity. In the latter a mazimal specific activity of ~ 800 umoles/ Pi/mg protein/hour is achieved. The two ion transporting tissues in the nauplii of the brine shrimp are the salt glands and gut tubes. Small scale isolation of the salt glands and dissection of minimal amounts of gut tubes re­vealed that these two tissues contain about 50% of the total Na, K-ATPase in the animal. The salt glands (~50 cells) contain 17% and the gut tubes about 43% of the enzyme. To obtain an adequate amount of enzyme for further study, large scale isolation is essential; the tissue must remain viable and the enzyme activity  should be maintained during the isolation procedure. This is achieved as shown in sections II and III, two of the results. The purity of the tissues is proven by microscopy. The specific activities in the total homogenate is 32 + 6.0 umoles Pi/mg protein /hour for the salt gland enzyme and 15 + 2.0 umoles Pi/mg protein/hour for the gut tube enzyme. In both cases, the activity is singnificantly higher than that of the kidney medulla enzyme.

Among the critical steps of the isolation procedure is the period of incubation at 37°C in the high phosphate medium (SGRM). The longer the duration of the incubation, the higher the yield of salt glands. Lowy  has shown that no salt glands are released in incubation of less than 6 hours. The maximum salt gland release occurs in around 12 hours of incubation. Beyond 12-14 hours, the number of salt glands released decreases rapidly to zero. However, attempts at using 10-12 hour periods for large scale isolation revealed that both viability and enzyme activity decreases significantly. It is reported in Materials and Methods that the optimal period for the incubation in SGR, at 37ْ C is found to be 8 hours. Another critical step with the large scale preparation is the time required for isolating the salt glands and gut tubes after naupliar fragmentation. It seems that some of the cells break during this process releasing proteolytic enzymes and other intracellular components which markedly decrease the viability of the Na,K-ATPAase in the salt glands and gut tubes. The nauplii are thus fragmented in small batches, and in 5 seconds. The slurry is immediately poured over a 142 um nylon mesh and the eluent directly passed through a 72 um mesh. This minimizes the exposure of salt glands and gut tubes to cellular fragments since all smaller fragments pass through the 72 um nylon mesh. All steps from there on are opimized to minimize enzyme inactivation during the purification. The process of collecting enough salt gland and gut tube enzyme for further purification requires finding on the gut enzyme . The change in relative abundance of the 2 subunits in different tissues, both in development and in response to hormones such as insulin may reflect a new mechanism for regulating intracellular Na and other ions which in turn may play a role in DNA replication, translation and transcription and hence in the control of growth, development and differentiation. The fact that absence of insulin alters the  relative abundance of the two α subunits in fate cells which is reversed by insulin administration, also suggests that the same sequence of events may play a role in the development of the long term complications seen in diabetes mellitus particularly the proliferative ones.

The other objective is to achieve differential labelling of membrane proteins and lipids, each with a different isotope. ³⁵S-methionine added to the ASW incorporates into a host of naupliar proteins including the α and β subunits. Continuous labelling experiments as well as the pulse chase studies indicate that the increased activity of the enzyme with development is consequent to increased synthesis. Sodium acetate 5-10 mM blocks significantly the uptake of tritiated amino acids into membrane lipids. An amino acid mixture 2.5 mM in each amino acid blocks the uptake of ³H-acetate into membrane proteins. Work in progress by J. Usta and K. Badjakian on the turnover rates of the various membrane lipid components will determine, from the rates of synthesis and degradation, the optimal period of precursor pulsing that will achieve maximal selevtive labelling. The ultimate objective of these studies is to achieve differential labelling of membrane proteins and lipids with tritium and deuterium in the hope of obtaining three dimensional images of the enzyme in its lipid core and under different ligand conditions, by neutron diffraction. The labelled subunits can be isolated by electroelution and column chromatography and reconstituted into lipid vesicles for functional and structural studies.