Ultrastructural localization of phosphatase activity in the guinea pig pineal gland by the cerium technique.

Abstract

Thiamine pyrophosphatase (TPPase), nucleoside diphosphatase (NDPase), and glucose-6-phosphatase (G-6-Pase) were localized by the cerium technique in guinea pig pinealocytes and compared with the corresponding lead technique. NDPase and TPPase were also compared at different pH values using the cerium technique. Vibratome sections of perfusion-fixed tissue were incubated with cerium chloride or lead nitrate. Substrates used were thiamine pyrophosphate (for TPPase), sodium inosine diphosphate (NDPase), and disodium glucose-6-phosphate (G-6-Pase). The 1-2 trans saccules of the Golgi apparatus showed TPPase and NDPase activity but none for G-6-Pase. The endoplasmic reticulum (ER) cisternae and perinuclear space had NDPase and G-6-Pase activity but not TPPase. The abluminal plasmalemma of endothelial cells and the plasmalemma of Schwann cells demonstrated TPPase and NDPase activity but the luminal plasmalemma of the endothelial cells and the plasmalemma of pinealocyte processes showed only NDPase activity. TPPase was active at all pH values tested, but NDPase was most active at pH values of 6.5 and 7.0. Lead phosphate precipitate was frequently seen in nuclei, perinuclear space, ER cisternae, and "synaptic" vesicles when lead was used as the capturing agent. These sites were usually not labeled when cerium was used.

Glucose‐6‐phosphatase activity of endoplasmic reticulum and Golgi apparatus in spermatocytes and spermatids of the rat: an electron microscopic cytochemical study

“…In spermatocytes, all ER cisternae displayed G6Pase activity similar to the ER reactivity observed in Leydig cells, Sertoli cells and other cell types [19,22,41]. In contrast, the ER of spermatids was not uniformly labeled with cerium since some of the ER cisternae distal to the Golgi apparatus were G6Pase-positive, while others were either slightly reactive or negative.…”

“…In addition, the consistency of the G6Pase activity in acidic, neutral or slightly alkaline media minimized the possibility raised by Tokunaga et al [41] that the reactivity observed in the Golgi apparatus may be due to acid phosphatases, which are known to be present in the Golgi apparatus of spermatids in particular [4,38,39]. Recent results obtained by Luo et al [22] on the ultrastructural localization of various phosphatases in cells of the pineal gland also tend to validate the use of the cerium technique in the cytochemical localization of phosphatases.…”

“…Whenever observed, the lead phosphate precipitate was spotty over the ER and Golgi apparatus, and, in addition, a fine nonspecific lead deposit was observed over the nucleus and cytoplasm of the various testicular cells. The utilization of cerium chloride as a capture agent instead of lead nitrste, originally recommended by Veenhuis et al [42], and utilized by some investigators thereafter [14,20,22,33], considerably improved the localization of G6Pase activity within testicular cells. Indeed, the consistency of the results obtained in several successive experiments, the uniformity of the distribution of the fine cerium precipitate over ER cisternae and some Golgi elements as well as the absence of non-specific dusty cerium deposits over the nucleus and cytoplasm of the testicular cell argued in favor of using cerium ions as capture agents.…”

Ultrastructural localization of activity of phosphatases by low temperature incubation of unfixed cryostat sections

“…Thiamine pyrophosphatase is generally considered to be a cytochemical marker for the trans side of the Golgi complex in many cell types (Cheetham et al 1971;Angermüller und Fahimi 1984;Roth et al 1985;Luo et al 1990). Our data demonstrate that FRP due to thiamine pyrophosphatase activity was not only present in trans side cisternae but also in medial and cis side cisternae of Golgi complexes in acinar cells of unfixed rat parotid glands (Fig.…”

“…For the detection of 5′-nucleotidase and thiamine pyrophosphatase activities in liver and parotid gland which were freshly fixed or fixed after freezing and thawing, the tissue blocks were preincubated in media containing 100 mM TRIS-maleate buffer (pH 7.2), 2 mM CeCl 3 , and 2 mM MgCl 2 or 100 mM TRIS-maleate buffer (pH 7.2), 2 mM CeCl 3 , and 2 mM MnCl 2 for 30 min at 22° C, respectively (Luo et al 1990), followed by incubation for 30 min at 37° C in complete incubation media in the presence of 1 mM AMP or 1 mM TPP, respectively. All steps were performed under gentle continuous shaking.…”