Proteolytic processing of pro-opiomelanocortin occurs in acidifying secretory granules of AtT-20 cells.

Using antibodies specific for pro-opiomelanocortin (POMC), amidated joining peptide (JP), and the prohormone convertase PC1, we showed immunocytochemically that PC1 in a corticotrophic tumor cell line, AtT-20, was co-localized either with POMC or with amidated JP in secretory granules, and also confirmed that POMC was cleaved mainly in secretory granules. Analysis using DAMP (3- [2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as the pH probe suggested a correlation between POMC processing and acidic pH in the secretory granules. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, completely inhibited POMC processing and caused constitutive secretion of the unprocessed precursor. By contrast, chloroquine, a weak base that is known to neutralize acidic organelles, was unable to inhibit POMC processing. Electron microscopic analysis revealed that, in AtT-20 cells treated with bafilomycin A1, the trans-Golgi cisternae were dilated and few secretory granules were present in the cytoplasm. These observations suggest that acidic pH provides a favorable environment for proteolytic processing of POMC by PC1 but is not required, and that integrity of the trans-Golgi network and sorting of POMC into secretory granules are important for POMC processing.

Comparative biochemical study of coated vesicles purified from cock brains.

Cock brain coated vesicles (CBCVs) were purified and compared with porcine brain coated vesicles (PBCVs) from several biochemical aspects. Clathrin heavy and light chains of CBCVs were immunologically similar to those of PBCVs. Coat proteins (CPs) of CBCVs behaved in almost the same manner as those of PBCVs for limited proteolysis. Dissociation of CPs from CBCVs by treatment with 10 mM Tris-Cl, pH 8.5, or 2M urea resembled that of CPs from PBCVs. pH dependency of dissociation of CPs from CBCVs was slightly different from those of PBCVs.

Isolation and characterization of cock brain coated vesicles in comparison with porcine brain coated vesicles.

1. Cock brain coated vesicles (CBCVs) were isolated and compared with porcine brain coated vesicles (PBCVs). 2. The fine structure of CBCVs was quite similar to that of PBCVs. 3. Size distribution of CBCVs showed a rather single population, whereas that of PBCVs seemed to consist at least two subpopulations. 4. CBCVs possessed proteins quite characteristic of coated vesicles (CVs). 5. The protein composition of CBCVs was very like that of PBCVs with the exception of clathrin light chain b. 6. A small difference in the electrophoretic mobility existed between CBCVs and PBCVs. 7. The density of CBCVs was slightly greater than that of PBCVs.

Purification and characterization of a molecular weight 100,000 coat protein from coated vesicles obtained from bovine brain.

A protein designated as a 100-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis was purified from coated vesicles obtained from bovine brain, with uncoated vesicles as starting material. Two gel filtration steps, one involving 0.5 M tris(hydroxymethyl)aminomethane, pH 8.0, buffer, and the other 0.01 M tris(hydroxymethyl)aminomethane, pH 8.0, and 3 M urea buffer, were employed. The purified protein has a native molecular weight of 114,000 as determined by sedimentation equilibrium analysis. Circular dichroism data showed that the protein has 28% helical structure, 29% beta-structure, and 15% beta-turns, and the rest is random coil. Addition of the purified protein to clathrin results in the polymerization of clathrin to homogeneous size baskets of sedimentation velocity 150 S. A scan of the Coomassie Blue stained electrophoresis gels of the polymerized baskets shows that, for every clathrin trimer, there is approximately one 100-kDa protein molecule.

Comparative biochemical study of alkaline phosphatase isozymes in fish, amphibians, reptiles, birds and mammals.

Alkaline phosphatases from the liver, kidney and intestine in various vertebrates were strongly inhibited by beryllium, 2-mercaptoethanol, potassium cyanide and EDTA. The enzymes showed various sensitivities to the inhibition by zinc and to heat denaturation at 56 degrees C for 5 min at pH 7.0. The liver and kidney enzymes showed higher sensitivity to the inhibition by L-homoarginine than by L-phenylalanine. The intestinal enzymes in higher vertebrates were more sensitive to the inhibition by L-phenylalanine than by L-homoarginine, whereas the intestinal ones in lower vertebrates showed quite similar sensitivities to both amino acids.

Effects of dibutyryl adenosine 3':5'-cyclic monophosphate and other agents on induction of alkaline phosphatase activity in monkey kidney cells.

The induction of alkaline phosphatase (ALP) by dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) was investigated in strain JTC-12 . P3 cells derived from monkey (Maccaca irus) kidney cortex. ALP activity was increased by Bt2cAMP in a dose-dependent manner, reaching a plateau at concentrations higher than 5 mM with the activity being about 4 times that of the controls. The concentration of Bt2cAMP required for half-maximal induction of ALP activity was about 0.8 mM. ALP activity was increased rapidly by Bt2cAMP for the first 5 days and then continued to increase gradually towards a plateau level. Removal of Bt2cAMP from the medium caused a rapid decrease in the activity, suggesting that the induction of ALP activity by Bt2cAMP is reversible. ALP activity was induced synergistically in the presence of 1 mM sodium butyrate together with Bt2cAMP at concentrations from 0.01 to 1 mM. It was also found that in the presence of 1 mM Bt2cAMP, sodium butyrate increased ALP activity in the same manner as Bt2cAMP did in the presence of 1 mM sodium butyrate. Although dexamethasone, a potent glucocorticoid, had no effect on ALP activity in control cells, the hormone suppressed the ALP activity induced by Bt2cAMP in a dose-dependent manner. At concentrations above 0.2 mM, two xanthine derivatives, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX), also inhibited the induction of ALP activity by 1 mM Bt2cAMP. Inhibitors of protein synthesis, cycloheximide (1.5 micrograms/ml) and pactamycin (10 micrograms/ml), as well as inhibitors of RNA synthesis, actinomycin D (2 micrograms/ml) and alpha-amanitin (50 micrograms/ml), suppressed the induction of ALP activity.

The effects of prostaglandin E2, parathyroid hormone, 1,25 dihydroxycholecalciferol, and cyclic nucleotide analogs on alkaline phosphatase activity in osteoblastic cells.

Clone MC3T3-E1 cells isolated from newborn mouse calvaria had the same type of alkaline phosphatase (ALP) as that found in adult mouse calvaria (the liver-bone-kidney type), as judged by polyacrylamide gel electrophoresis as well as by heat lability and amino acid inactivation. The effects of prostaglandin E2 (PGE2), parathyroid hormone (PTH), 1,25 dihydroxycholecalciferol [1,25(OH)2D3], and adenosine-3',5'-cyclic monophosphate (cAMP) analogs on ALP were investigated. PGE2 and 1,25(OH)2D3 increased ALP activity in dose-related manner with a maximal effect at concentrations of 10 ng/ml and 40 pg/ml, respectively. N6,O2-dibutyryl adenosine-3',5'-cyclic monophosphate (DBcAMP) also induced an increase in ALP activity in a dose-related fashion with a maximal effect at a concentration of 0.5 mM which was 2.2-fold over that of the controls. Induced ALP was of the "liver-bone-kidney" type. Antinomycin D and cycloheximide inhibited the increase in ALP activity induced by DBcAMP. The level of ALP was elevated by 8-bromo-adenosine-3',5'-cyclic monophosphate and theophylline, but not by N6,O2-dibutyryl guanosine-3',5'-cyclic monophosphate and sodium butyrate. Moreover, PGE2 dramatically increased the level of cAMP in the cells with a maximal effect at a concentration of 10 ng/ml, indicating that PGE2 and DBcAMP induce an increase of ALP activity in clone MC3T3-E1 cells by increasing the cAMP level; PTH did not affect enzyme activity and cAMP level in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioaffinity chromatography of thyrotropin using immobilized concanavalin A.

The behavior of crude preparations of whale and bovine thyrotropins was studied on an affinity column packed with concanavalin A-Sepharose. Only a small portion of the proteins applied was adsorbed to the column and eluted quantitatively with 0.5 M methyl-alpha-D-glucoside or -mannoside. Immunoreactive as well as hormonally active thyrotropin was recovered exclusively in the absorbed fraction. The usefulness of this chromatographic procedure for the group separation of pituitary glycoprotein hormones is discussed.