A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

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In the hematopoietic system a pluripotent stem cell generates precursors for lymphoid and myeloid lineages. Proenkephalinderived peptides were previously detected in differentiated lymphoid cells. We have studied whether the proenkephalin system is expressed in a typical differentiated cell of the myeloid lineage, the neutrophil. Human peripheral polymorphonuclear cells contain and release proenkephalin-derived peptides. The opioid portion of proenkephalin (met-enkephalincontaining peptides) was incompletely processed, resulting in the absence of low molecular weight products. The nonopioid synenkephalin (proenkephalin 1-70) molecule was completely processed to a 1.0-kD peptide derived from the COOH-terminal. This molecule was characterized in neutrophils by biochemical and immunocytochemical methods. The chemotactic peptide FMLP and the calcium ionophore A23187 induced the release of the proenkephalin-derived peptides, and this effect was potentiated by cytochalasin B. The materials secreted were similar to those present in the cell, although in the supernatant a higher proportion corresponded to more processed products. The 1.0-kD peptide was detected in human, bovine, and rat neutrophils, but the chromatographic pattern of synenkephalin-derived peptides suggests a differential posttranslational processing among species. These findings demonstrate the existence of the proenkephalin system in human neutrophils and the production and release of a novel 1.0-kD peptide derived from the synenkephalin molecule. The presence of opioid peptides in neutrophils suggests their participation in the inflammatory process, including a local analgesic effect. (J.

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The subtilisin-like enzyme PC1 (also known as PC3) cleaves the neuropeptide precursor proopiomelanocortin at paired basic residues in transfection experiments, thus providing evidence for a critical role in precursor processing. While mRNA for this enzyme is highly enriched in neuroendocrine tissues, little is known about the tissue and subcellular distribution of the PC1 protein. This study used immunocytochemical techniques to investigate the anatomical distribution of PC1, both alone and compared to met-enkephalin (MET-enk), in AtT-20 pituicytes transfected with proenkephalin cDNA. A high density of PC1 immunostaining was observed in a small region adjacent to the nucleus and in the tips of the processes of these cells. Dual-staining immunocytochemistry of whole cells illustrated that both PC1 and MET-enk immunoreactivity were present in the tips, but PC1 was concentrated in a region adjacent to the nucleus while MET-enk punctate staining was dispersed throughout the soma. This codistribution was confirmed in semithin sections of dual-stained cells cut at 1-1.5 µm through the thickness of the cells. PC1 staining resembled that of TGN38, a marker for the trans-Golgi network. When PC1 immunocytochemistry was performed in cells that were pretreated with brefeldin A, a drug that redistributes the proximal Golgi compartments to the endoplasmic reticulum, there was a complete disruption of the defined locus of PC1 immunoreactivity. Taken together, our data indicate that (1) PC1 is concentrated in a region of the cell body resembling the trans-Golgi network and (2) both the enzyme and the processed peptide are transported to the tips of the processes. The observation of abundant PC1 within the Golgi complex supports the notion that proteolytic processing of neuropeptide precursors may be initiated in the Golgi apparatus rather than occurring solely within secretory granules.