Yeast aspartic protease 3 (YAP3p), a basic-residue specific proprotein processing enzyme, was shown to be a membrane-associated protease. The membrane association of YAP3p was demonstrated to be through a glycophosphatidylinositol anchor situated in the carboxy terminus of the enzyme. Carboxy-terminal truncation of YAP3p by 37 amino acids resulted in secretion of YAP3p into the growth medium. Western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two secreted forms of YAP3p with apparent molecular masses of approximately 180 and approximately 90 kDa. YAP3p has an isoelectric point of approximately 4.5 as determined by isoelectric focusing gel electrophoresis. Treatment of YAP3p with endoglycosidase H reduced the size of both forms of the protein to approximately 65 kDa, consistent with the presence of 10 potential N-linked glycosylation sites in the deduced amino acid sequence of this protein. Removal of the N-linked sugars did not affect the enzymatic activity of YAP3p. Analysis of the effect of temperature on the stability and the rate of enzymatic activity of YAP3p showed that the enzyme retained 100% of its activity when incubated for 1 h at 37 degrees C, while incubation at 50 degrees C for 1 h resulted in approximately 80% loss of activity. The dependence of activity on temperature demonstrated a calculated Q10 of 1.95.

The presence of [arginine] vasopressin (AVP) mRNA and AVP immunoreactivity in pituicytes of the neural lobe (NL) of intact and pituitary stalk-transected rats, with and without osmotic stimulation, was examined. AVP mRNA was analyzed by Northern blotting, as well as by in situ hybridization in combination with immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) as a marker for pituicytes. In intact rats, a poly(A) tail-truncated 0.62-kb AVP mRNA was detected in the NL and was found to increase 10-fold with 7 days of continuous salt loading. Morphological analysis of the NL of 7-day salt-loaded rats revealed the presence of AVP mRNA in a significant number of GFAPpositive pituicytes in the NL and in areas most probably containing nerve fibers. Eight days after pituitary stalk transection the NL AVP mRNA diminished in animals given water to drink, whereas in those given 2% saline for 18 h followed by 6 h of water, a treatment repeated on 6 successive days beginning 2 days after surgery, the 0.62-kb AVP mRNA was present. The AVP mRNA in the pituitary stalk-transected, salt-loaded rats showed an exclusive cellular distribution in the NL, indicative of localization in pituicytes. Immunoelectron microscopy showed the presence of AVP immunoreactivity in a subpopulation of pituicytes 7 and 10 days after pituitary stalk transection in salt-loaded animals, when almost all AVP fibers had disappeared from the NL. These data show that a subset of pituicytes in the NL is activated to synthesize AVP mRNA and AVP in response to osmotic stimulation.The [arginine] vasopressin (AVP) gene is abundantly expressed in magnocellular neurons of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) in the hypothalamus (1). However, recent studies have shown the presence of AVP mRNA in the neural lobe (NL) of the pituitary as well, where the axons of these neurons terminate. This NL mRNA is shorter than that present in the hypothalamus due to a truncated poly(A) tail and is up-regulated during osmotic stimulation (2-5). After disconnection of the hypothalamoneurohypophysial tract by electrically damaging the ventromedial hypothalamic area, the AVP mRNA disappeared from the NL (5). This latter observation, together with the detection of AVP mRNA at the electron microscopic level in a subset of axonal swellings in the median eminence and NL (6), has led investigators to propose that the NL AVP mRNA is derived from the hypothalamic magnocellular neurons and is axonally transported to the NL (5, 6). Furthermore, the poly(A) tail of this AVP mRNA appears to be truncated during axonal transport (7). The existing data, however, do not fully exclude the possibility that some of the NL AVP mRNA is expressed in pituicytes.In this study, we have carried out in situ hybridization histochemistry in combination with immunocytochemistry, using a glial fibrillary acidic protein (GFAP) antibody as a marker for the pituicytes (8, 9) to examine the expression of AVP mRNA in these astrocytes in the NL. Studies were conducte...

The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full‐length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full‐length 86‐kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl‐terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane‐associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86‐kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus‐truncated 64‐kDa form.

The relationships between delta sleep-inducing peptide (DSIP) and GnRH immunoreactivity within the guinea pig median eminence are investigated by light and electron microscopic immunocytochemistry. Indirect immunofluorescence and elution-restaining experiments show that at the light microscopic level the distribution patterns of DSIP and GnRH immunoreactivity are indistinguishable. This suggested the possible coexistence of both immunoreactivities within the same fibers and neurosecretory endings. At the electron microscopic level, a preembedding double-immunolabeling technique using both indirect immunoperoxidase and immunogold methods, clearly indicate that DSIP and GnRH immunoreactivity are frequently colocalized within single secretory granules. In addition DSIP/GnRH immunoreactive nerve endings were also observed often in close proximity to tanycyte elements. Taken together, the present results provide for the first time ultrastructural evidence for the presence of DSIP immunoreactivity and demonstrate that DSIP and GnRH immunoreactivities may be coexpressed within the same neuronal elements in the median eminence.