Altered biosynthesis and secretion of pro-opiomelanocortin in the intermediate and anterior pituitary of carboxypeptidase E-deficient, Cpe(fat)/ Cpe(fat)mice.

The biosynthesis and secretion of pro-opiomelanocortin (POMC) was examined in the pituitary of Cpe(fat)/ Cpe(fat)mice, which are deficient in carboxypeptidase E, a sorting receptor for the regulated secretory pathway (Cool D R, Normant E, Shen F S, et al. Cell 1997; 83: 73-83). Dopamine inhibited forskolin-stimulated accumulation of cAMP in the intermediate lobe of Cpe(fat)/ Cpe(fat)mice, showing that their dopamine receptors were fully functional. This result indicates that the elevated, dopamine-insensitive POMC secretion previously observed in the intermediate pituitary of Cpe(fat)/ Cpe(fat)mice was constitutive, rather than due to defective dopamine receptors. Concomitant with the increase in POMC secretion was a twofold increase in POMC mRNA levels and [(35)S]-methionine incorporation into POMC. In the anterior pituitary of Cpe(fat)/ Cpe(fat)mice, a 1.6-fold increase in basal release of POMC was accompanied by a similar increase in [(35)S]-methionine incorporation into POMC, although POMC mRNA levels were unchanged. Thus, the intermediate and anterior pituitary of Cpe(fat)/ Cpe(fat)mice compensate for the constitutive secretion of POMC by upregulating biosynthesis.

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in cpefat mice associated with a carboxypeptidase E mutation.

Cpefat mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia-diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in Cpefat mice. In Cpefat mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K+ stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of Cpefat mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of Cpefat mice.

Regulation of HIV-1 gag protein subcellular targeting by protein kinase C.

The human immunodeficiency virus type 1 internal structural protein precursor, p55, and its corresponding matrix proteolytic fragment, p17, are phosphorylated at Ser111 by protein kinase C. COS-7 cells transfected with plasmids encoding either the wild-type or Ser111-->Ala mutated human immunodeficiency virus type 1 gag gene matrix domain proteins were treated with phorbol 12-myristate 13-acetate (PMA), and the phosphorylation of the expressed p17 proteins was examined by radioimmunoprecipitation, SDS-polyacrylamide gel electrophoresis, and autoradiography. PMA treatment of transfected cells resulted in a 4-5-fold increase in wild-type p17 (but not mutated p17) phosphorylation; however, mutated p17 exhibited a low basal level of phosphorylation that was not affected by PMA, suggesting that additional sites were phosphorylated. PMA treatment of cells expressing wild-type p17 produced a dramatic shift in the localization of p17 from the cytosol to the membrane fraction within 8-15 min, followed by a slow quantitative dissociation of p17 back into the cytosol by 90 min. The cytosol-to-membrane translocation was dependent on N-myristoylated p17 since cells expressing p17 with a Gly2-->Ala mutation did not localize to the membrane. PMA also failed to induce the translocation of fully N-myristoylated Ser111-->Ala p17, suggesting that p17 phosphorylation at Ser111 was responsible for membrane association. This conclusion was confirmed by the finding of phosphorylated wild-type p17 in the membrane fraction only after PMA treatment. These results suggest that a "myristoyl-protein switch" regulates the reversible membrane targeting of p17 by protein kinase C-mediated phosphorylation. This signal may provide a mechanism for the cellular regulation of virus development through modulation of gag protein-related developmental steps such as capsid targeting, assembly, encapsidation, budding, and maturation.

Characterization of the okra mucilage by interaction with Gal, GalNAc and GlcNAc specific lectins.

A bio-active polysaccharide, which was the major component of the extract of the common okra, Hibiscus esculentus, was isolated from the extract by precipitation with ethanol between 28.5 to 45%. According to a previous report (Whistler, R.L. and Conrad, H.E. (1954) J. Am. Chem. Soc. 76, 1673-1674), this polysaccharide contains the Gal alpha 1-->4Gal sequence, which is the ligand for the uropathogenic Escherichia coli and toxic lectins. Analysis of the binding property of the okra polysaccharide by precipitin assay with Gal, GalNAc and GlcNAc specific lectins showed that this okra mucilage reacted best with Mistletoe toxic lectin-I (ML-I) and precipitated over 80% of the ML-I nitrogen (5.1 micrograms N) added. It also precipitated well with Abrus precatorius (APA), Momordica charantia (MCA) and Ricinus communis (RCA1) agglutinins, but poorly with other lectins. The results obtained suggest that this polysaccharide is a valuable reagent to differentiate Gal specific lectins from the GalNAc and/or GlcNAc specific series.

Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells.

The biosynthesis of the prohormone convertase PC2 was studied in Chinese hamster ovary cells stably transfected with PC2 cDNA (CHO/PC2) and in rat insulinoma cells (Rin5f). The major form of PC2 synthesized by CHO/PC2 cells was a 75-kDa protein corresponding to proPC2; this protein was retained intracellularly for 2-4 h following synthesis, suggesting prolonged intracellular residence. In contrast, the major form of PC2 within Rin cells initially exhibited a molecular mass of 72 kDa and was then progressively converted to a 64-kDa species. This 64-kDa species, which required 1-2 h to be released, was the major PC2 form detectable in Rin cell medium. Calcium-dependent benzyloxycarbonyl-Arg-Ser-Lys-Arg-aminomethylcoumarin cleaving activity was found in spent Rin cell medium; this activity could be immunoprecipitated with a carboxyl-terminal PC2 antibody, but not with preimmune serum. In neither cell line did intracellular PC2 become endoglycosidase H-resistant over time. PC2 released from Rin cells was also endoglycosidase H-sensitive. Microsequencing and endoglycosidase H results indicate that 75-kDa CHO cell PC2 and 72-kDa Rin cell PC2 both represent proPC2. We speculate that (a) PC2 undergoes unusual glycosylation, which may be related to its slow release from cells, and (b) the 64-kDa molecule detectable in spent Rin cell medium represents the enzymatically active form of PC2.

A putative processing enzyme for proenkephalin in bovine adrenal chromaffin granule membranes. Purification and properties.

A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.

Purification and assay of opioid activity of low molecular weight enkephalin-immunoreactive peptides generated by peptic digestion of rat plasma proteins.

A low molecular weight Methionine enkephalin-immunoreactive peptide (MEIP) and two Leucine-enkephalin immunoreactive peptides (LEIP) generated by peptic digestion of rat plasma were purified through gel filtration followed by five sequential reverse phase HPLC gradients in different solvent systems. Binding experiments of these peptides to opioid receptors of rat brains were performed. The two LEIPs were able to inhibit binding of [3H]naloxone to opioid receptors in rat brain membranes. No inhibition was found with the MEIP (which represented the only MEIP present in the low molecular weight fraction of pepsin-digested rat plasma). Sequencing revealed that the MEIP is a six residue peptide with the following sequence: Gly-Glu-Tyr-Gly-Phe-Gln. This sequence corresponds to that of residues 422-427 of rat serum albumin.

Characterization of enkephalin-immunoreactive peptides generated by peptic digestion of rat plasma proteins.

Met-Enkephalin-immunoreactive peptides (MEIP) and Leu-enkephalin-immunoreactive peptides (LEIP), generated from plasma proteins after digestion with pepsin, were characterized with gel filtration, reverse phase HPLC, and isoelectric focusing. Pooled rat plasma was found to generate 170 pmol LEIP and 76 pmol MEIP/ml plasma. Two peaks of enkephalin (enk)-immunoreactive peptides (EIP) were observed after gel filtration of plasma digests; the early eluting peak (which contained 90% of the total LEIP and 75% of the total MEIP) eluted close to, but not in, the void volume of the column, whereas the elution volume of a later eluting peak (which contained 25% of the total MEIP and about 5% of the total LEIP) was close to that of authentic enk. Five peaks of EIP with pI values of 3.7, 5.8, 7.3, 8.0, and 9.4, were observed after isoelectric focusing of plasma digests; with the exception of the pI 3.7 peak, all of these immunoreactive species could be detected with greater efficiency using the Leu-enk RIA. Peptic digestion of rat serum albumin generated a MEIP with the same pI and HPLC retention time as the plasma pI 3.7 MEIP. No other EIP could be generated from rat serum albumin. No immunoreactive peptides were found which coeluted with synthetic Met-enk or its sulfoxide on HPLC; however, a portion of low mol wt LEIP was eluted with the retention time of authentic Leu-enk. With certain exceptions, these results support and extend the original findings of Singer and Carraway concerning the ability of pepsin to generate extremely high concentrations of EIPs from plasma protein(s).