The changes of cellular Ca²⁺ homeostasis via modulation of the Ca²⁺-transporting systems could be the origin of a number of functional disorders in organisms. Functioning of membrane transporting systems is regulated through the amino acid residues in active centres of their molecules. SH-groups are the most sensitive part of the membrane Ca²⁺-transporting molecules and are identified as an important part of the functional domains of Ca²⁺-ATPases and Na⁺ÐCa²⁺ exchanger. Because the types and properties of SH-groups of Ca²⁺, Mg²⁺-ATPases of secretory cell membrane are still unknown, the aim of our work was to identify and describe functional SH-groups in SERCA and PMCA molecules. To reveal the functional importance of SH-groups, their properties and type, we used p-chlormercuribenzoate (PCMB). The study was performed using post-nucleus, post-mitochondria microsome fractions obtained from isolated secretory cells of rat submandibular salivary gland by a series of consequent centrifugations. Using specific inhibitors (oubain and thapsigargin) and specific marker tests (digitonin treatment) allowed us to identify the microsome fraction as a mixture of plasma membrane and endoplasmic reticulum vesicles, most of which are inside-out. Specific activities of Ca²⁺, Mg²⁺-ATPases in the microsome fraction was measured using the Fiske-Subarrow method. We show that the membrane fraction is rich with active Ca²⁺, Mg²⁺-ATPase molecules. The maximal initial rate of ATP hydrolysis is 0.29 ± 0.06 mM P_i min^-1 (1 mg protein)^-1. The activity of the thapsigargin-insensitive fraction of total Ca²⁺, Mg²⁺-ATPase (PMCA) was 0.11 ± 0.01 mM P_i min^-1 (1 mg protein)^-1 and thapsigargin-sensitive (SERCA) 0.4 ± 0.01 mM P_i min^-1 (1 mg protein)^-1; data are expressed as means ± S.E.M., n = 8 in all experiments.

We have shown that PCMB (1Ð1000 mM) has an inhibitory effect on the Ca²⁺, Mg²⁺-ATPases. Its apparent inhibitory constant I₅₀ for PMCA and SERCA are 245 ± 47 and 52 ± 6 mM, respectively. At concentrations higher than 400 mM, PMCA and SERCA were completely blocked. Such data allowed us to speculate about the availability of functionally important SH-groups in the catalytic centre of PMCA and SERCA. In order to understand the mechanism of Ca²⁺, Mg²⁺-ATPase inhibition by PCMB, we used dithiothreitol (DTT), a powerful chelator of SH-groups. Incubation of vesicles with DTT caused increase of PMCA and SERCA activities (by 22 ± 8 and 48 ± 12 %) allowing us to suppose that DTT could facilitate ATP access to the catalytic centre of molecule. The most prominent restoring effect of DTT on SERCA and PMCA inhibited by PCMB was 45 ± 8 and 32 ± 11 %, respectively. That is why we suppose that the functioning of PMCA is predominantly regulated by masked SH-groups. Summarizing all data obtained we can conclude that PMCA and SERCA have high reactive SH-groups, which comprise an important part of the catalytic domain of their molecules and regulate their functioning.

All procedures accord with current local guidelines.