Regulation of S-adenosylmethionine decarboxylase in L1210 leukemia cells. Studies using an irreversible inhibitor of the enzyme

A potent irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, S-(5'-adenosyl)-methylthio-2-aminooxyethane (AdoMeSaoe), was used to study the regulatory control of this key enzyme in the polyamine biosynthetic pathway. Treatment of L1210 cells with the inhibitor completely eradicated the growth-induced rise in AdoMet decarboxylase activity, resulting in a marked decrease in cellular content of spermidine and spermine. The putrescine content, on the other hand, was greatly elevated. Although no detectable AdoMet decarboxylase activity was found in the L1210 cells after treatment with AdoMeSaoe, the cells contained 50-fold higher amounts of AdoMet decarboxylase protein, compared to untreated cells during exponential growth. Part of this increase was shown to be due to elevated synthesis of the enzyme. This stimulation appeared to be related to the decrease in cellular spermidine and spermine content, since addition of either one of the polyamines counteracted the rise in AdoMet decarboxylase synthesis. The synthesis rate was determined by immunoprecipitation of labeled enzyme after a short pulse with [35S]methionine. In addition to a protein that co-migrated with pure rat AdoMet decarboxylase (Mr approximately 32,000), the antibody precipitated a somewhat larger labeled protein (Mr approximately 37,000) that most likely represents the proenzyme form. Treatment of the L1210 cells with AdoMetSaoe also gave rise to a marked stabilization of the decarboxylase which contributed to the increase in its cellular protein content. Addition of spermidine did not significantly affect this stabilization, whereas the addition of spermine reduced the half-life of the enzyme to almost that of the control cells.     

Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus

Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.     

Detection of proenzyme form of S-adenosylmethionine decarboxylase in extracts from rat prostate

Previous work in which the synthesis of S-adenosylmethionine decarboxylase was studied by translation of its mRNA indicated that it was formed as a proenzyme having a M.W. of about 37,000 that was cleaved to form the enzyme sub-unit of M.W. 32,000 in a putrescine-stimulated reaction. The extent to which the proenzyme accumulates in vivo and is affected by the putrescine concentration was studied by subjecting prostate extracts to Western immunoblotting procedures. The proenzyme form was readily detectable in control prostates (about 4% of the total) and this proportion was increased to 25% when the rats were pretreated for 3 days with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. Conversely, it was decreased to almost undetectable levels after treatment with methylglyoxal bis(guanylhydrazone). These results indicate that the processing of the proenzyme form of S-adenosylmethionine decarboxylase is regulated by the cellular putrescine concentration. This conversion provides another step at which polyamine biosynthesis may be controlled.     

Regulation of S-adenosylmethionine decarboxylase activity in rat liver and prostate

Two methods were used for the quantitation of S-adenosylmethionine decarboxylase protein. The first involved titrating the active site of the enzyme by reduction of the Schiff base between 3H-decarboxylated S-adenosylmethionine and the pyruvate prosthetic group with sodium cyanoborohydride. The second method was radioimmunoassay with rabbit antiserum which was used to determine the total immunoreactive enzyme protein. It was found that the increased S-adenosylmethionine decarboxylase activity produced in rat prostate by treatment with alpha-difluoromethylornithine and in both prostate and liver by methylglyoxal bis(guanylhydrazone) were due entirely to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either method) remained constant in rats treated with the drugs. Treatment with 2% alpha-difluoromethylornithine in the drinking water for 3 days increased prostatic S-adenosylmethionine decarboxylase protein by 5-fold. A substantial part, but not all, of this increase could be accounted for by a slowing of the rate of degradation of the enzyme. The half-life for loss of activity and titratable protein after inhibition of protein synthesis by cycloheximide was increased from 35 to 108 min by treatment with alpha-difluoromethylornithine. However, the half-life for loss of immunoreactive protein which was considerably longer was only increased from 139 to 213 min. The molecular weight of the S-adenosylmethionine decarboxylase subunit determined by immunoblotting was 32,000, and no smaller immunoreactive fragments were detected. These results indicate that spermidine depletion produced by alpha-difluoromethylornithine affects the degradation of S-adenosylmethionine decarboxylase at an early step involving the loss of the active site without substantial breakdown of the protein.     

Structural characterization of the proteins encoded by adenovirus early region 2A

Proteins encoded by adenovirus type 2 and type 5 early region 2A isolated from infected HeLa cells were compared to translation products of E2A-specific messenger RNA in a reticulocyte cell-free system and in Xenopus oocytes. The main cell-free translation product is a 72,000 Mr polypeptide which in HeLa cells as well as in Xenopus oocytes is converted into a 75,000 Mr phosphoprotein capable of binding to single-stranded DNA. Some minor proteins are proteolytic cleavage products of the major protein. In the cell-free system, three E2A polypeptides, 32,000, 37,000 and 44,000 Mr, are translated from minor polyadenylated mRNA species that can be separated from the major mRNA. Synthesis of all E2A polypeptides in vitro is inhibited by cap-analogs. The 44,000 Mr protein is also synthesized in Xenopus oocytes. Tryptic peptide maps of [35S]methionine-labeled E2A proteins were constructed using high pressure liquid chromatography and the position of the methionyl residues within each peptide was determined by amino acid sequencing procedures. This information and the DNA sequence of the adenovirus 5 E2A gene published by Kruijer et al. (1981) were used to align the peptides and to construct a map of the E2A proteins. Our data demonstrate that the major 75,000 Mr protein is coded for by a leftward reading frame of 529 amino acid residues located between 62 and 66 map units. The data also map six sites as targets for proteolytic enzymes. The minor E2A translation products have the same carboxy terminus as the major protein. The initiation codons of the 44,000, 37,000 and 32,000 Mr polypeptides probably correspond to amino acids 170, 243 or 244 and 290 of the major protein. Some functional properties of the major E2A protein are shared by the minor proteins and thus could be mapped. Major sites of phosphorylation, the region involved in binding to single-stranded DNA and the antigenic regions recognized by immune sera are located between amino acid residues 50 to 120, 170 to 470 and 170 to 240, respectively.     

Studies of functional domains of the regulatory subunit from cAMP-dependent protein kinase isozyme I

Homogenous regulatory subunit from rabbit skeletal muscle cAMP-dependent protein kinase (isozyme I) was partially hydrolyzed with low (1 g/1300 g) or high (1 g/6 g) concentrations of trypsin. After treatment with low trypsin two main peptides (Mr = 35,000 and 12,000) were produced. The cAMP-binding activity (2 mol cAMP/mol of subunit monomer) was recovered in the monomeric Mr = 35,000 peptide. The ability of either fragment to inhibit catalytic subunit activity was lost. Treatment of the regulatory subunit with a high concentration of trypsin yielded three main fragments (Mr = 32,000, 16,000, and 6,000) which could be resolved by Sephadex G-75 and purified further on DEAE-cellulose columns. One of the peptides (Mr = 32,000) bound 2 mol cAMP/mol fragment. The Mr = 16,000 fragment was very labile and bound cAMP with an undetermined stoichiometry. Cyclic AMP dissociation curves for the native regulatory subunit and its Mr = 32,000 component were similar and suggested the presence of two nonidentical binding sites in each monomer. Using the same procedure, the Mr = 16,000 fragment or homogenous cGMP-dependent protein kinase appeared to contain a single type of binding site. Purified Mr = 32,000 fragment was readily converted to the Mr = 16,000 fragment using high trypsin as assessed by protein bands on SDS-disc gels or by following transfer of radioactivity from Mr = 32,000 peptide covalently labeled with 8-N3-[32P] cAMP to radiolabeled Mr = 16,000 fragment. The smallest regulatory subunit fragment (Mr = 6,000) did not bind cAMP, but was dimeric and could be part of the dimerization domain in the native protein. A model is presented to explain the possible structural-functional relationships of the regulatory subunit.     

Inhibition of translation of mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase by polyamines

The effect of spermidine and spermine on the translation of the mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase was studied using a reticulocyte lysate system and specific antisera to precipitate these proteins. It was found that the synthesis of these key enzymes in the biosynthesis of polyamines was much more strongly inhibited by the addition of polyamines than was either total protein synthesis or the synthesis of albumin. Translation of the mRNA for S-adenosylmethionine decarboxylase was maximal in a lysate which had been substantially freed from polyamines by gel filtration. Addition of 80 microM spermine had no significant effect on total protein synthesis and stimulated albumin synthesis but reduced the production of S-adenosylmethionine decarboxylase by 76%. Similarly, addition of 0.8 mM spermidine reduced the synthesis of S-adenosylmethionine decarboxylase by 82% while albumin and total protein synthesis were similar to that found in the gel-filtered lysate. Translation of ornithine decarboxylase mRNA was greater in the gel-filtered lysate than in the control lysate but synthesis of ornithine decarboxylase was stimulated slightly by low concentrations of polyamines and was maximal at 0.2 mM spermidine or 20 microM spermine. Higher concentrations were strongly inhibitory with a 70% reduction occurring at 0.8 mM spermidine or 150 microM spermine. Further experiments in which both polyamines were added together confirmed that the synthesis of ornithine and S-adenosylmethionine decarboxylases were much more sensitive to inhibition by polyamines than protein synthesis as a whole. These results indicate that an important part of the regulation of polyamine biosynthesis by polyamines is due to a direct inhibitory effect of the polyamines on the translation of mRNA for these biosynthetic enzymes.     

Calmodulin binding proteins in rat liver mitochondria

Calmodulin binding proteins have been found in submitochondrial fractions obtained from highly purified rat liver mitochondria. The matrix fraction contains two major calmodulin binding proteins: one, having Mr of 145,000, apparently is carbamoyl-phosphate synthetase. Another has a Mr of 58,000 and has not been associated with enzyme activities. A major calmodulin binding protein of unknown function and having Mr of 32,000 has been found in the Triton X-100 solubilizate of the inner membrane. Minor amounts of two calmodulin binding proteins having Mr of about 37,000 and 56,000 have been found in the outer membrane.     

Identification of the types of insulin-like growth factor-binding proteins that are secreted by muscle cells in vitro

We have previously shown that muscle cells secrete insulin-like growth factor-binding proteins. In the present study, BC3H-1 cells were shown to secrete one binding protein of Mr 32,000, whereas L6 cells secreted two binding proteins of Mr 31,000 and 24,000, as determined by ligand blotting. Subconfluent proliferating L6 cells secrete more of the Mr 24,000 binding protein, relative to the Mr 31,000 form. In contrast, differentiated L6 myotubes secreted similar quantities of the two forms. Insulin-like growth factor I preferentially stimulated secretion of the Mr 31,000 versus the Mr 24,000 binding protein from L6 cells and caused an increase in the secretion of the Mr 32,000 binding protein from BC3H-1 cells. The Mr 31,000 binding protein from L6 cells had a greater affinity for insulin-like growth factor II compared with insulin-like growth factor I, as did the Mr 32,000 binding protein of BC3H-1 cells. In contrast, the Mr 24,000 binding protein of L6 cells preferred insulin-like growth factor I. Neither porcine insulin nor relaxin competed for 125I-IGF-I binding. In conclusion, these muscle cell lines secrete only one or two forms of insulin-like growth factor-binding proteins. L6 cell differentiation is associated with a relative increase in the secretion of the Mr 31,000 binding protein compared with the Mr 24,000 form. Insulin-like growth factor I stimulates the secretion of its own binding proteins from muscle cells, and this may be an important mechanism for modulating cellular responsiveness to this growth factor.     

Proteins encoded by the long terminal repeat region of mouse mammary tumor virus: identification by hybrid-selected translation.

The long terminal repeat (LTR) region of mouse mammary tumor virus (MMTV) is known to contain an open reading frame of sufficient length to code for a protein of 36,000 Mr. The coding capacity of the 3' sequences of MMTV genomic RNA has been demonstrated by in vitro translation studies, which have reported the synthesis of four related proteins: p36, p24, p21, and p18. These proteins are overlapping translation products of the same open reading frame, with the smaller ones initiating at internal methionine codons. From the predicted amino acid sequence of the LTR protein, we have selected a region likely to be antigenic, obtained a synthetic peptide of that region, and raised antiserum to the peptide. The antipeptide serum specifically immunoprecipitated all four proteins from in vitro translated genomic 3' MMTV RNA, plus an additional one of 32,000 Mr. Published sequence data of MMRV LTRs show an internal AUG codon at a position which could initiate a protein of 32,000 Mr. The three smaller in vitro translation products (p24, p21, and p18) were consistently synthesized in much greater amounts than the p36 or p32 protein. The relative amount of each in vitro synthesized protein from genomic MMTV RNA could be predicted and was in good agreement with the postulated effect of flanking nucleotides on the efficiency of the respective AUG initiation codon. Polyadenylated RNAs, isolated from various mouse tissues, were selected by hybridization to plasmid DNA containing MMTV LTR sequences immobilized on nitrocellulose. In vitro translation of hybrid-selected mRNAs isolated from BALB/c mouse lactating mammary glands and carcinogen-induced mammary tumors, followed by immunoprecipitation with antipeptide serum, revealed that only one polypeptide was synthesized by the MMTV LTR-specific mRNA, the 36,000 Mr species.     

The speEspeD operon of Escherichia coli. Formation and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C. W., Tabor, H., and Xie, Q.-W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6040-6044). We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme. Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit. The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity. Both subunits are present in the purified enzyme. These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D. L., and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822).     

Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., J?nne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.     

Identification of canine pulmonary surfactant-associated glycoprotein A precursors

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000-28,000 daltons; glycoprotein A2, 32,000-34,000 daltons; and glycoprotein A3, 37,000-38,000 daltons; pH at isoelectric point (pI) 4.5-5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8-5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000-34,000 daltons, pI 4.8-5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000-28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.     

Quantitation of S-adenosylmethionine decarboxylase protein

A method for the specific labeling of the active site of S-adenosylmethionine decarboxylase was developed. The method consisted of incubating cell extracts with 3H-decarboxylated S-adenosylmethionine and sodium cyanoborohydride in the presence of a spermidine synthase inhibitor. Under these conditions, S-adenosylmethionine decarboxylase was labeled specifically and stoichiometrically. This procedure was used (a) to establish that the subunit molecular weight of S-adenosylmethionine decarboxylase from rat liver, prostate, and psoas and from mouse SV-3T3 cells was 32 000, (b) to titrate the number of active molecules of S-adenosylmethionine decarboxylase in various cell extracts, and (c) to provide a high specific activity labeled preparation of S-adenosylmethionine decarboxylase for use in radioimmunoassay of this enzyme. Competitive radioimmunoassays using this labeled antigen had a sensitivity such that 3 fmol (0.1 ng) of enzyme protein could be quantitated. The rapid loss of S-adenosylmethionine decarboxylase which occurred when SV-3T3 cells were exposed to exogenous polyamines was shown to be due to a rapid decline in the amount of enzyme protein measured both by titration of the active site and by radioimmunoassay.     

Escherichia coli S-adenosylmethionine decarboxylase. Subunit structure, reductive amination, and NH2-terminal sequences

S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.     

Isolation of a cDNA clone encoding S-adenosylmethionine decarboxylase. Expression of the gene in mitogen-activated lymphocytes

S-Adenosylmethionine decarboxylase was purified from bovine liver and digested with endopeptidase Lys-C; the resulting peptides were chromatographically separated. Peptides containing either methionine or tryptophan were subjected to sequence analysis. An oligonucleotide mixture of 48 sequences, which was 17 nucleotides in length, was synthesized based on one of these peptide sequences. This synthetic oligonucleotide mixture was labeled and used to screen a bovine cDNA library in phage lambda gt11. A clone was identified which contained a 1350-nucleotide insert. This insert contained nucleotide sequences coding for amino acid sequences of two of the peptides that were analyzed, thus proving that this cDNA clone codes for S-adenosylmethionine decarboxylase. A subcloned fragment from the coding region of the cDNA was used as a probe to analyze the expression of this gene in mitogen-activated lymphocytes. Northern blots revealed two message species of 2.4 and 3.6 kilobases in length. Both mRNAs were coordinately expressed and were present in polysomes. The levels of these mRNAs increased approximately 4-fold by 9 h after activation of the cells. The magnitude of the increase in these messages is to be compared with an 8- to 10-fold increase in the rate of synthesis of the protein. The apparent increase in translational efficiency of this message upon lymphocyte activation was confirmed by analyzing polysomes from these cells. In resting lymphocytes, the average size of polysomes containing mRNA coding for S-adenosylmethionine decarboxylase was 1.4 ribosomes per mRNA, and this value increased to 2.7 in stimulated cells. Thus, it appears that the increase in translational efficiency of this mRNA arises from an elevated rate of translational initiation, leading to more ribosomes per polysome encoding this particular message. This is not a general effect on the expression of all proteins, since there is no change in the translational efficiency of cytoplasmic actin upon activation of lymphocytes.     

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells.

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.     

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.