Parital characterization of the rat anti-encephalitogenic protein (RSCP).

A protein antigenically similar to the anti-encephalitogenic bovine spinal cord protein (BSCP) was detected in saline extracts of rat nervous tissues by immunodiffusion analyses using a rabbit anti-BSCP serum. Rat SCP (RSCP) appears to be evenly distributed throughout all parts of the rat nervous system and occurs also in the thymus, thyroid, and adrenal glands. Although immunodiffusion analyses indicated that RSCP shares some antigenic sites with BSCP, anti-RSCP sera reacted only with RSCP, indicating that the major immunogenic determinants of the RSCP are peculiar to the rat and differ from the immunogenic determinants of human, monkey, rabbit, guinea pig, or bovine SCP. Immunoelectrophoretic analyses of concentrated pastes of rat brain (RB) or rat spinal cord (RSC) in agar at pH 8.6 revealed that RSCP occurs in two molecular forms having the electrophoretic mobilities of a serum beta-globulin and a serum gamma-globulin, respectively. However, gamma-RSCP is the predominant component of extracts of brain or spinal cord. Gamma-RSCP was isolated from RB and RSC by a procedure which involved: a) extraction with 0.05 M ammonium acetate buffer, pH 4.0; b) batch absorption of impurities on CM-52 cellulose; c) batch absorption of RSCP on SP-Sephadex, pH 3.5; d) elution of RSCP from SP-Sephadex, pH 5.5; and finally, e) gel filtration on Sephadex G-50 superfine. Purified gamma-RSCP formed one band when analyzed by polyacrylamide electrophoresis in acid gels containing 8 M urea. In contrast, two bands were always present when gamma-RSCP from brain or spinal cord were subjected to SDS-polyacrylamide electrophoresis in 15% gels. The larger of the two components of brain gamma-RSCP had a m.w. of 12,400 daltons, whereas the two components of spinal cord gamma-RSCP were smaller. The molecular sizes of brain RSCP and spinal cord RSCP was estimated by gel filtration chromatography to be 12,400 daltons. The amino acid compositions of gamma-RSCP prepared from RB or RSC were similar except that gamma-RSCP from RSC contained twice as much half-cystine and a slightly higher proportion of basic amino acid than gamma-RSCP from RB.     

Isolation and characterization of glycogen branching enzyme from rabbit liver

Glycogen branching enzyme was isolated from rabbit liver. The highly purified enzyme shows a monomer molecular weight of 71 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and apparent molecular weights of 93 000 by sucrose density gradient sedimentation and 52 000 by gel-exclusion chromatography on Sephacryl S-300. No glucosamine, mannosamine, galactosamine, or sialic acid was detected in the protein. An amino acid analysis is reported. The spectrum of branching enzyme is that of a simple polypeptide, with A1%280nm = 24.6. Highly purified branching enzyme consists of several closely related active enzyme forms that can be resolved by isoelectric focusing in polyacrylamide gel. The major species of pI 5.7 is flanked by less abundant forms of pI 5.6 and 5.8. Seemingly identical enzyme forms are observed in crude extracts of rabbit liver, skeletal muscle, brain, and heart, although the absolute and relative concentrations vary among the tissues. Branching enzyme apparently does not exhibit tissue-specific isoenzymes.     

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.     

ACTIVATION, INHIBITION AND AGGREGATION OF CHOLINE ACETYLTRANSFERASE (EC 2.3.1.6)

—Mercuric chloride, silver acetate and cupric sulphate (0·1 mm ) completely inhibited purified choline acetyltransferase from bovine caudate nuclei. At the same concentration cadmium chloride and zinc acetate gave a 50 per cent inhibition. Potassium and sodium salts more than doubled the enzymatic activity while creatinine hydrochloride more than tripled it. Guanidine hydrochloride was less effective than creatinine hydrochloride but more effective than KCl and NaCl. Sodium chloride and creatinine hydrochloride had a synergistic effect on the enzyme. When ammonium sulphate was used to fractionate the choline acetyltransferase that had been extracted from bovine caudate nuclei, the enzyme aggregated into different molecular sizes as determined by exclusion chromatography on Bio-gel A-1·5 m. The molecular weight of the largest aggregate was at least 10⁶ daltons. The initial tissue extract contained only one molecular species of ChAc as did a partially purified preparation in which ammonium sulphate was not used in the purification.     

Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

Synthesis and amino acid composition of basic proteins in mammalian sperm nuclei.

The basic chromosomal proteins (SCP) of human, mouse, rabbit and guinea pig sperm nuclei were characterized by polyacrylamide gel electrophoresis and amino acid analysis. Spermatozoa were decapitated with 1% SDS and the nuclei recovered by density gradient centrifugation. Examination by Nomarski and electron microscopy revealed the nuclei to be intact and 99% pure. The basic proteins were extracted from nuclei, aminoethylated and purified by ion exchange chromatography and gel filtration chromatography.The SCP of human, rabbit and guinea pig gave single protein bands with similar mobilities when subjected to polyacrylamide gel electrophoresis. In contrast, aminoethylated mouse SCP consisted of two proteins, SCP·AE₁ and SCP·AE₂, which had different electrophoretic mobilities. The SCP of these mammalian species were characteristically rich in arginine (47–54.4%) and cysteine (7.7–12.2%). Major differences existed in the amino acid compositions of these proteins. Mouse and human SCP were rich in histidine (12.2 and 7.7%, respectively) and guinea pig was high in tyrosine (11.7%) and phenylalanine (3.5%). Valine was detected only in rabbit SCP and proline in human and guinea pig. Aspartic acid, methionine and tryptophan were not detected in all four species. Studies on the incorporation of [³H]arginine into mouse SCP demonstrated that these basic proteins are synthesized during the terminal stages of spermatogenesis and are subsequently conserved.     

Isolation,purification and some properties of a lectin from the winged bean (Psophocarpus tetragonolobus)

We have isolated by affinity chromatography a lectin from the seeds of the winged bean (Psophocrapus tetragonolobus) which agglutinated human (group A, B and O), sheep and rabbit, but not mouse erythrocytes. A molecular weight of 41,000 was obtained from gel filtration, and on sodium dodecyl sulphate polyacrylamide gel electrophoresis a single polypeptide chain of molecular weight 35,000 was seen both before and after reduction. Isoelectric focussing of the lectin on polyacrylamide gel gave a single band with a calculated isoelectric point of 4.0. The lectin was found to be rich in acidic amino acids; cysteine was not detected. Carbohydrate analysis revealed no covalently bound sugars.Abbreviations PBS phosphate-buffered saline - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - WBL winged bean lectin - HPLC high performance liquid chromatography     

Purification and characterization of a lectin from the mushroom, Flammulina veltipes

A lectin was purified to homogeneity from the mushroom, Flammulina veltipes, by zinc acetate treatment and CM-cellulose column chromatography. Its molecular weight was estimated to be 20,000 by gel filtration and polyacrylamide gel electrophoresis. The lectin does not contain carbohydrate, half-cystine, methionine, or histidine. On gel filtration sith Sepharose 6B in the presence of 6M guanidine-HCl, the purified lectin dissociated into two nonidentical subunits, FVA-L (molecular weight, 12,000) and FVA-S (8,000). The hemagglutinating activity was retained only in the FVA-L subunit. The lectin is mitogenic with respect to mouse spleen lymphocytes.     

Purification of a brain-specific astroglial protein by immunoaffinity chromatography

An immunoaffinity chromatography procedure for the isolation of bovine glial fibrillary acidic (GFA) protein is described. Degraded GFA protein isolated by hydroxyapatite chromatography from human spinal cord was used to prepare the antiserum. The immunoglogulin G fraction of the antiserum was covalently linked to CNBr-activated Sepharose, and columns of the immuno-affinity gel were used to adsorb bovine GFA protein from brain extracts. Elution was accomplished with a solution of 1 m acetic acid, 5 m urea, 0.8 m sodium chloride, pH 2.5. The yield of about 0.5 mg of highly purified protein/g of cerebral white matter could be increased to 1.5 mg/g of tissue by lowering the ionic strength of the extracting buffer from 50 mm to 1 mm sodium phosphate. Isolation in the presence of EDTA prevented the formation of an oxidation product migrating as a dimer of the monomeric species on SDS-polyacrylamide gel electrophoresis.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Subunit structure of AMP-deaminase from chicken and rabbit skeletal muscle.

The AMP-deaminases from chicken and rabbit muscle have been investigated by techniques which include sedimentation equilibrium, sodium dodecyl sulfate gel electrophoresis, amino acid analysis, NH2- and COOH-terminal analyses, and tryptic peptide mapping. The molecular weights of the native chicken (276,000) and rabbit (271,000) enzymes obtained by sedimentation equilibrium studies are in good agreement with values of 276,000 (chicken) and 275,000 (rabbit) calculated from amino acid analyses. The enzymes were reduced, carboxymethylated, and treated with either maleic or succinic anhydride in the presence of 6 M guanidine hydrochloride. Sodium dodecyl sulfate gel electrophoresis of the chemically modified enzymes resulted in a single electrophoretic species having an apparent molecular weight of 85,000. This observation is consistent with previous studies on the nonacylated enzymes and suggests that the muscle AMP-deaminases from chicken and rabbit do not contain noncovalent linkages which are readily disrupted by a large increase in negative charge. NH2-terminal analyses by the method of Stark and Amyth as well as the dansyl technique, indicate that the NH2-terminal positions of these enzymes are blocked. The enzymes are also resistant to digestion with carboxypeptidases A or B (or both) in the presence of sodium dodecyl sulfate. The most distinctive feature of the amino acid compositions of both the chicken and rabbit AMP-deaminases is the presende of eight half-cystine residues per 69,000 g of protein. Tryptic digests of the S-14C-carboxymethylated proteins were fractionated by ion exchange chromatography and high voltage electrophoresis. Six and five radioactiviely labeled peptides were detected in the electrophoretograms of the chicken and rabbit enzymes, respectively. This observation and the number of ninhydrinposition spots, together with the physical data on the molecular weights of the native enzymes and their subunits, suggest that the AMP-deaminases from chidken and rabbit muscle consist of four identical or very similar polypeptide chains.     

Immunological and biochemical evidence for atrial-like isomyosin in thyrotoxic rabbit ventricle

Immunological, structural and enzymatic characteristics of atrial and ventricular myosin from euthyroid rabbits were analyzed and compared with ventricular myosin from hyperthyroid animals. (1) Specific antibodies against bovine atrial myosin were found to react selectively in double-immunodiffusion and enzyme immunoassay with both rabbit atrial myosin and ventricular myosin from thyroxine-treated animals. These specific anti-bovine atrial myosin antibodies reacted with the heavy chains of thyrotoxic ventricular myosin when examined by enzyme immunoassay combined with SDS-polyacrylamide gel electrophoresis. (2) In one-dimensional SDS-polyacrylamide gel electrophoresis, no difference could be demonstrated in the light chain pattern of ventricular myosin from euthyroid and hyperthyroid rabbit hearts. One-dimensional analysis of myosin heavy chains after chymotryptic digestion in the presence of SDS showed significant differences between the two ventricular myosins. Also, the peptide maps from atrial myosin resembled the pattern of peptides found with ventricular heavy chains from hyperthyroid rabbits. The steady state rate, the alkali stability and the pH sensitivity of Ca2+-ATPase activity of thyrotoxic ventricular myosin were very similar to those of atrial myosin. (3) These results provide direct immunochemical and biochemical evidence for the existence of an atrial-like isomyosin in thyrotoxic rabbit ventricles.     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

Isolation and characterization of rabbit H of the alternative complement pathway

Rabbit factor H, a control protein of the alternative complement pathway, was isolated from rabbit serum by polyethylene glycol precipitation, DEAE-Sephacel chromatography, and gel chromatography on Sephadex G200. The protein migrated as a single-chain polypeptide with a molecular weight of 160,000 on sodium dodecyl sulfate-gel electrophoresis with Laemmli's buffer system, but hardly migrated into the gel with Fairbanks' buffer system. Physical and chemical properties of rabbit H were similar to those of human H, except that fragments produced by limited tryptic digestion from rabbit H had different molecular sizes from those produced from human H. Significant species-specificity was observed in the functional activity of factor H; activation of the alternative complement pathway was inhibited more efficiently with homologous H than with heterologous H. In contrast, factor H inhibited the hemolysis of homologous erythrocytes less than that of heterologous erythrocytes.     

Neutral microprotein, a novel human plasma and urinary protein associated with a yellow-brown chromophore. Isolation from protein HC preparations and partial characterization

An apparently novel human plasma and urinary protein of low molecular weight was isolated from several highly purified preparations of protein HC by gel chromatography and high voltage electrophoresis with a yield of about 8 mg/g. The protein has a molecular weight of about 20,000, neutral electrophoretic mobility at pH 6.5 and a high content of half-cystine. It is associated with a yellow-brown chromophore like protein HC and could be demonstrated in all investigated preparations of isolated human, rabbit and guinea-pig protein HC and alpha 1-microglobulin.     

IMMUNOCHEMICAL AND BIOCHEMICAL STUDIES DEMONSTRATING THE IDENTITY OF A BOVINE SPINAL CORD PROTEIN (SCP) AND A BASIC PROTEIN OF BOVINE PERIPHERAL MYELIN (BF)

A protein extracted from bovine peripheral myelin (BF) and a protein extracted from bovine spinal cord (SCP) have been shown to be identical: the proteins cross-react immunochemicaliy with each other but not with highly purified CNS myelin basic protein. Neither BF nor SCP have anti-encephalitogenic activity. Their electrophoretic behavior is the same at three different pH values. Their apparent molecular weight by sodium dodecyl sulfate-gel electrophoresis is 13,800 ± 550. The amino acid compositions of the proteins are essentially identical. BF and SCP each contain 2 cysteine residues and have valine at the C terminus. The 23 major tryptic peptides are identical on peptide maps. Circular dichroic analyses yield essentially identical curves, which, when computed by best-fit curve analysis, indicate that each has 0%α helix and a large percentage of β structure.     

Calcium binding of arterial tropomyosin: involvement in the thin filament regulation of smooth muscle

Bovine aortic tropomyosin has been isolated by DEAE-Sepharose chromatography following isoelectric precipitation and ammonium sulfate fractionation. A single polypeptide [Mr 36 000 on a sodium dodecyl sulfate (SDS)-polyacrylamide gel] was obtained under different electrophoretic conditions. The amino acid composition of bovine tropomyosin was very similar to that of rabbit skeletal muscle; the amino-terminal residue is blocked. The molecular weight of the native tropomyosin (76 000), which is twice that calculated from the SDS-polyacrylamide gel, suggests that the molecule is a dimer. The diffusion coefficient of 3.4 X 10(-7) cm2 s-1 and the frictional coefficient of 1.7 indicate that the molecule is asymmetric. Comparative high-pressure liquid chromatography peptide mapping of rabbit skeletal and bovine aortic tropomyosins shows primary structure variation. Bovine aortic tropomyosin binds calcium under physiological conditions of pH and ionic strength (22 mol of Ca2+/mol of tropomyosin with a Kd of 1.4 mM). Such a property is not shared by skeletal tropomyosin. In low Mg2+ concentration, both skeletal and aortic actin activations of the skeletal myosin ATPase activity are calcium independent. Addition of aortic tropomyosin to a hybrid actomyosin (aortic actin, skeletal myosin) yields an enhancement of the actin activation of the myosin ATPase activity, but the addition of skeletal tropomyosin yields a decrease of this activity. However, both the enhancement and decrease are calcium dependent. Addition of skeletal or aortic tropomyosin to an actomyosin system, where both actin and myosin come from skeletal muscle, yields only an enhancement of the actin activation of the myosin ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural features of bovine fetuin revealed from analysis of the primary translation product: anomalous behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is due largely to peptide and not solely to carbohydrate

Native bovine fetuin is a major alpha 1-glycoprotein in the serum and cerebrospinal fluid of fetal calves. We previously reported (Johnson, W.V., and Heath, E.C. (1986) Biochemistry 25, 5518-5525) the purification of the primary translation product for fetuin (prefetuin) from a rabbit reticulocyte cell-free translation system and showed that prefetuin contains an 18 amino acid signal peptide. Here we report that although the apparent sodium dodecyl sulfate (SDS) gel molecular weights of fetuin and prefetuin are 64,000 and 49,000, respectively, when analyzed by gel filtration chromatography under denaturing and reducing conditions, molecular weight values of 48,000 and 40,000 were found for native fetuin and the nonglycosylated prefetuin, respectively. These molecular weight values are in agreement with those expected on the basis of the sedimentation diffusion data of Spiro (Spiro, R.G. (1960) J. Biol. Chem. 235, 2860-2869) and indicate that the polypeptide moiety makes a major contribution toward the anomalous SDS gel electrophoretic mobility of fetuin. Therefore, the carbohydrate moiety is not solely responsible for this property. Edman degradation of [35S]methionine-labeled prefetuin indicated that the N-terminal residue is the only methionine present in prefetuin; native fetuin lacks methionine. Additionally, hydroxylamine cleavage of an Asn-Gly bond in prefetuin localized one of the N-linked carbohydrate side chains to the middle of the polypeptide chain of native fetuin.     

Multiple forms of endothelial cell growth factor. Rapid isolation and biological and chemical characterization

Endothelial cell growth factor (ECGF) can be rapidly purified from bovine brain to high specific activity using heparin-Sepharose affinity chromatography. Purification of the mitogen by this method results in relatively high yields of the polypeptide (10 to 100 micrograms/kg of tissue) with biological activity on murine and human endothelial cells in the picogram range. The product obtained is a mixture of two single-chain polypeptides with apparent molecular weights of 17,000 (alpha-ECGF) and 20,000 (beta-ECGF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two forms of ECGF can be separated by either NaCl gradient elution from heparin-Sepharose or reversed-phase high pressure liquid chromatography. The two polypeptides are related on the basis of similar: amino acid compositions, affinity for heparin-Sepharose, cyanogen bromide and trypsin-derived cleavage products, and biological activity. Furthermore, the cyanogen bromide fragments derived from the two forms of ECGF also possess similar amino acid compositions and mobilities on sodium dodecyl sulfate gels. These data suggest that there are at least two discrete molecular forms of ECGF in bovine brain and that these two molecules are structurally related.     

Studies on the synthesis of sterol carrier protein-2 in rat adrenocortical cells in monolayer culture. Regulation by ACTH and dibutyryl cyclic 3',5'-AMP

The effects of ACTH or dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of sterol carrier protein-2 (SCP2) have been studied in rat adrenocortical cells in monolayer culture. Radiolabeling of total cellular proteins with [35S]methionine and immunoprecipitation with antibodies directed against rat liver SCP2, followed by polyacrylamide gel electrophoresis and fluorography, showed a 3-4-fold increase in the rate of synthesis of SCP2 in cells treated for 48 h with ACTH (1 microM) or Bt2cAMP (0.1 mM). The induction of SCP2 synthesis depended upon the concentrations of ACTH or Bt2cAMP with an ED50 of 8 and 100 nM, respectively, and increased linearly with time between 12 and 48 h of treatment. Immunoprecipitation of SCP2 synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from cells treated with ACTH or Bt2cAMP revealed increased synthesis of SCP2 compared to RNA from control cells. The immunoprecipitable rat adrenal SCP2, synthesized in a cell-free translation system, showed mobility corresponding to Mr of 14,400 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was clearly larger than immunodetectable SCP2 synthesized in cultured adrenal cells (Mr = 11,300). The electrophoretic mobilities of rat liver SCP2 synthesized in cultured cells and in a cell-free translation system were the same as the respective forms from rat adrenal. It is concluded that the synthesis of SCP2 in rat adrenocortical cells is induced by ACTH and that the induction is mediated by cAMP and may involve increased levels of translatable mRNA encoding a higher molecular weight precursor form of SCP2, which presumably undergoes post-translational processing yielding the mature form.