Ectonucleotidase activities in Sertoli cells from immature rats

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 + or - 6 and 21 + or - 2 nmol Pi mg-1 min-1 for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 + or - 2 nmol Pi mg-1 min-1 for AMP and 1.52 + or - 0.13 nmol adenosine mg-1 min-1, respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules

The study of the catalytic sites of enzymes using fluorescent compounds

The use of fluorescent compounds can be a valuable tool to probe the active site of enzymes. Several examples of this approach are discussed, particularly the use of pyridoxal phosphate analogs. The study of protein-protein interactions by means of fluorescent-labeled proteins is also analyzed

The H+ -ATP synthase: a biochemical challenge

ATP is a high energy compound that living cells utilize for driving most of their endergonic reactions. Directly or indirectly, ATP yields energy through the splitting of its terminal pyrophosphate bond. In cells, the ATP synthase of energy transducing membranes is responsible for forming from ADP and phosphate most of the ATP that cells need for survival and reproduction. The question of how the enzyme catalyzes ATP synthesis has been addressed by numerous workers for over thirty years. A fundamental discovery was that the enzyme is localized in membranes, and that the energy for ATP formation derives from electrochemical gradients built up by enzymes that catalyze electron transfer and that are localized in those membranes. However, the molecular events that take place in the H+ -ATP synthase during the transformation of the energy of electrochemical gradients into the chemical energy of ATP have not been entirely unveiled. Studies of its structure have shown that the H+ -ATP synthase is one of the most complex enzymes discovered. It has a H+ conducting multisubunit pathway and a multisubunit complex where the catalytic events in ATP synthesis take place. Moreover, it is an enzyme that is regulated by numerous and different factors, i.e., adenine nucleotides, electrochemical H+ gradients and protein-protein interactions. Studies on the mechanisms of energy transduction have shown that synthesis of ATP at the catalytic site of the enzyme is a spontaneous process; this indicates that depending on the environment ATP may be a high or a low energy compound. Thus, even though the enzyme presents many unknowns, it continues to be a source of fundamental and unsuspected aspects of basic biochemistry.