Comparison of the Molecular Forms of the Kex2/Subtilisin‐Like Serine Proteases SPC2, SPC3, and Furin in Neuroendocrine Secretory Vesicles Reveals Differences in Carboxyl‐Terminus Truncation and Membrane Association

Abstract

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.

Purification and Characterization of Serine Proteases That Exhibit Caspase-Like Activity and Are Associated with Programmed Cell Death in Avena sativa

“…They could be retained in the ER where they await processing and secretion. The Kexin family of subtilisin-like Ser proteases is known to autocatalytically remove their pro-domain in the ER before continuing in the secretory pathway (Gluschankof and Fuller, 1994;Hill et al, 1995). A mutation in the Kex2 protease that inhibits removal of the prodomain stimulates accumulation of proKex2 in the ER (Gluschankof and Fuller, 1994).…”

Cellular colocalization and coregulation between hypothalamic pro-TRH and prohormone convertases in hypothyroidism

“…These antibodies were raised against either a PC1/3-glutathione fusion protein or a PC2-glutathione fusion protein (14) and generously provided by Dr. Nigel Birch (University of Auckland). After being rinsed with PBS, sections were incubated with Biotin-SP-conjugated AffiniPure Fab Fragment Donkey Anti-Rabbit IgG (H ϩ L; Jackson ImmunoResearch, West Grove, PA) at a 1:400 dilution in 0.5% blocking reagent (TSA kit) for 60 min, followed by streptavidin-HRP (TSA kit) 1:100 for 30 min, and finally incubated in Fluorophore Tyramide Amplification Reagent (supplied in TSA kit) at a dilution of 1:50 for 10 min.…”

Novel Early Target Genes of Parathyroid Hormone-Related Peptide in Chondrocytes

“…, PC1/3 or PC2 [both raised in rabbits against either a PC1-glutathione fusion protein or a PC2-glutathione fusion protein(25), provided by Dr. Nigel Birch (University of Auckland, Auckland, Australia) that recognizes proPC1/3 and active PC1/3 as well as proPC2 and active PC2; all diluted to 1:400]. Slides were incubated with primary antibodies for 60 min.…”