Coelution of the type II holoenzyme form of cAMP-dependent protein kinase with regulatory subunits of the type I form of cAMP-dependent protein kinase

The types and subunit composition of cAMP-dependent protein kinases in soluble rat ovarian extracts were investigated. Results demonstrated that three peaks of cAMP-dependent kinase activity could be resolved using DEAE-cellulose chromatography. Based on the sedimentation of cAMP-dependent protein kinase and regulatory subunits using sucrose density gradient centrifugation, identification of 8-N3[32P]cAMP labeled RI and RII in DEAE-cellulose column and sucrose gradient fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Scatchard analysis of the cAMP-stimulated activation of the eluted peaks of kinase activity, the following conclusions were drawn regarding the composition of the three peaks of cAMP-dependent protein kinase activity: peak 1, eluting with less than or equal to 0.05 M potassium phosphate, consisted of the type I form of cAMP-dependent protein kinase; peak 2, eluting with 0.065-0.11 M potassium phosphate, consisted of free RI and a type II tetrameric holoenzyme; peak 3, eluting with 0.125 M potassium phosphate, consisted of an apparent RIIC trimer, followed by the elution with 0.15 M potassium phosphate of free RII. The regulatory subunits were confirmed as authentic RI and RII based upon their molecular weights and autophosphorylation characteristics. The more basic elution of the type II holoenzyme with free RI was not attributable to the ionic properties of the regulatory subunits, based upon the isoelectric points of photolabeled RI and RII and upon the elution location from DEAE-cellulose of RI and RII on dissociation from their respective holoenzymes by cAMP. This is the first report of a type II holoenzyme eluting in low salt fractions with free RI, and of the presence of an apparent RIIC trimer in a soluble tissue extract.     

Separation of two HMM-S1 species from white muscle myosin

Heavy meromyosin subfragment 1 was resolved by chromatography on DEAE-cellulose into two fractions characterized by the nature of the alkali light chains present. It was shown that even in an HMM-S1 preparation with an extensive fragmentation of the heavy chain a polyacrylamide gel electrophoresis analysis differentiates alkali light chains among the light fragmentation components. A non-fragmented HMM-S1 was obtained from a papain digest of myofibrils and the chromatographic analysis supplied further evidence of the separation of the two species of HMM-S1 present in rabbit white muscle myosin.     

Purification of Prorennin mRNA and Its Translation in Vitro

Prorennin-specific messenger ribonucleic acid (mRNA) has been purified by a combination of sizing techniques, including Sepharose 2B chromatography and sucrose density gradient centrifugation, and affinity chromatography with poly (U)-Sepharose, from total nucleic acid extracted from dry ice-pulverized, fourth stomach of a calf. This mRNA bound to poly (U)-Sepharose, indicating that it contained a poly (A) sequence. The total translation product in the mRNA-dependent wheat germ system, upon addition of this mRNA, was identified as authentic prorennin by gel electrophoresis. The molecular weight of this mRNA was about 3.5 × 10⁵ as determined by gel electrophoresis. These results indicate that the synthesis of prorennin is directed by this mRNA 1,020 nucleotides in length and requires the full coding capacity of the molecule.     

The purification and molecular properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa.

Neurospora crassa contains three isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which are inhibited by tyrosine, tryptophan and phenylalanine respectively, and it was estimated that the relative proportions of the total activity were 54%, 14% and 32% respectively. The tryptophan-sensitive isoenzyme was purified to homogeneity as judged by polyacrylamide-gel electrophoresis and ultracentrifugation. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes were only partially purified. The three isoenzymes were completely separated from each other, however, and can be distinguished by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34 and polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the tryptophan-sensitive isoenzyme contained one type of subunit of molecular weight 52000. The molecular weight of the native enzyme was found to be 200000 by sedimentation-equilibrium centrifugation, indicating that the enzyme is a tetramer, and the results of cross-linking and gel-filtration studies were in agreement with this conclusion.     

Purification and properties of pyruvate kinase from the hepatopancreas of Carcinus maenas.

Pyruvatekinase from the hepatopancreas of the common shore crab, Carcinus maenas, was purified to a specific activity of 240 units/mg of protein in the assay conditions described. 2. In one method of purification the enzymic activity could be resolved into two fractions after chromatography on DEAE-cellulose. Fructose 1, 6-diphosphate was able to effect the conversion of one form (peak 1) into the second (peak 2). 3. In the presence of a saturating concentration of fructose 1, 6-diphosphate both forms of the enzyme were kinetically similar. 4. Polyacrylamide-gel electrophoresis of the enzyme 1 day after preparation showed a single protein band. On storage at least three protein bands became visible, all of which were associated with pyruvate kinase activity. 5. Chromatography of the enzyme on Sephadex G-200 indicated a mol.wt. of 247000, but in the presence of fructose 1, 6-diphosphate the elution volume of the enzyme increased corresponding to a mol.wt. of 193000. 6 Dissociation of the enzyme in sodium dodecyl sulphate and 2-mercaptoethanol followed by polyacrylamide-gel electrophoresis produced one major protein band with a mol.wt. of 55000.     

Separation and quantification of muropeptides with high-performance liquid chromatography

About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.     

Microheterogeneity of alpha and beta subunit of tubulin from microtubules of starfish (Asterias amurensis) sperm flagella

Reduced and alkylated tubulin, obtained by aqueous extraction of an acetone powder of axonemes of starfish sperm flagella, was separated into two peaks of protein by column chromatography on urea/hydroxylapatite with a linear gradient of sodium phosphate (2 mm to 250 mm, pH 6.5). Sodium dodecyl sulfate/ polyacrylamide gel electrophoresis indicated that the first peak mainly consisted of α-subunit, while the second was β-subunit of tubulin. Furthermore, when analyzed by gel isoelectric focusing, α and β-subunit were resolved into four distinct bands, respectively. These bands appeared between the pH range of 5.5 to 6.0 and never overlapped.     

Component A2 of the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum.

Component A2 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum has been purified 370-fold by liquid chromatography. Homogeneity was obtained by anaerobic preparative polyacrylamide gel electrophoresis. Component A2 is a colorless, air-stable protein consisting of a single polypeptide as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The relative molecular mass of the native protein was determined by high-performance, size exclusion chromatography to be Mr 52,000; on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a value of Mr 59,000 was obtained. When cell extract was subjected to N6-ATP-agarose affinity chromatography the methylcoenzyme M methylreductase system was resolved into two fractions; one of them was component A2. This work provides a new operational definition for component A2, i.e., its characteristic chromatographic behavior on N6-ATP-agarose. However, its functional definition is its ability to reconstitute the methylreductase activity with components A1, A3, and C. Several attempts to assign a role to component A2 are reported.     

Purification of creatine kinase from beef heart mitochondria]

53-fold purified creatine kinase is isolated from beef heart mitochondria by phosphate buffer extraction followed by chromatography on DEAE-cellulose and KM-cellulose and preparative electrophoresis in phosphate buffer density gradient. The purified enzyme was homogenous under electrophoresis in agarose gel and moved to cathode. The enzyme did not enter into separating gel under disc electrophoresis in conditions for the separation of neutral anc acid proteins, while under conditions for separating alkaline proteins it produced five fractions. The stability of creatine kinase under storage considerably decreased after the purification.     

Stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in mouse uterine epithelial cells by oestradiol-17 beta.

Two alpha-D-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) produced by Aspergillus tamarii were purified from the mycelial extract by a procedure including chromatography on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. Each of these enzymes showed a single protein band corresponding to the alpha-D-galactosidase activity when examined by polyacrylamide-gel electrophoresis. They catalysed the hydrolysis of o-nitrophenyl alpha-D-galactoside, melibiose, raffinose and stachyose, but did not attack the galactomannans. Their Mr values were respectively 265000 +/- 5000 and 254000 +/- 5000 by the method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate in each case showed a single protein band, with Mr 88000 and 77500 respectively. The purified enzymes contained carbohydrate, consisting of N-acetylglucosamine, mannose, glucose and galactose in the estimated molar proportions of 1:9:5:8 in alpha-galactosidase I.     

Further purification of epidermal growth factor by high-performance liquid chromatography

Epidermal growth factor (EGF) purified by the method of Savage and Cohen (J. Biol. Chem.247, 7601–7611 (1972) using DEAE-cellulose chromatography as the final purification step was further resolved into two major uv-absorbing components by reverse-phase high-performance liquid chromatography (HPLC). Both components, referred to as α-EGF and β-EGF, competed with ¹²⁵I-labeled EGF for the EGF receptor, induced premature eye opening in neonatal mice, and had an amino acid composition similar to that published by Savage et al. (J. Biol. Chem.247, 7612–7621 (1972). β-EGF migrated slightly faster than α-EGF during sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. α-EGF was fourfold more potent than β-EGF and was 10-fold more potent than DEAE-purified EGF in stimulating DNA synthesis in quiescent Rat-1 cells. HPLC purification of EGF can replace the DEAE-cellulose chromatography step currently used and produces a more potent and less heterogeneous EGF species.     

Analysis of human alpha-thrombin by hydrophobic interaction high-performance liquid chromatography

The major active form of human thrombin, alpha-thrombin, was analyzed by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). The chromatographic system included a polymeric phenyl column and elution was performed by a gradient, 2-0M sodium chloride (5-20 min). Total analysis time was 30 min per injection. By this method, a good resolution between alpha-thrombin and the proteolytically modified thrombin forms, beta- and gamma-thrombin, was obtained. In addition, the thrombin preforms, prothrombin, prethrombin 1, and prethrombin 2, were also resolved from alpha-thrombin in the system. The results from the HIC method were compared to those obtained from non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By this high-resolution chromatographic method, the rapid analysis of purified alpha-thrombin is possible.     

Purification and properties of four species of lysyl oxidase from bovine aorta

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.     

Rat haemoglobin heterogeneity. Two structurally distinct alpha chains and functional behaviour of selected components.

Six haemoglobins were separated analytically from haemolysates of adult Wistar rats (Rattus norvegicus) by cellulose acetate electrophoresis and preparatively by DEAE-cellulose chromatography. The globin chains were separated from unfractionated haemolysates by CM-cellulose chromatography by using a non-linear formic acid-pyridine gradient followed by CM-cellulose chromatography in 8M-urea by using a gradient of increasing Na+ concentration in phosphate buffer, pH 6.7. Two alpha chains and three non-alpha chains were identified. Chains isolated from purified haemoglobins were correlated with chains isolated from unfractionated haemolysates by electrophoresis on urea-starch gels to make presumptive assignments of the subunit composition of the six haemoglobin tetramers. Partial amino acid sequences were determined for the major and minor alpha chains. The oxygen equilibria of two of the major haemoglobin components and of the unfractionated haemolysate were examined at pH 7.5 and 8.0. The two purified haemoglobins exhibited similar oxygen affinities; the haemolysate, however, had a lower oxygen affinity than either of the two purified haemoglobins. Both the haemolysate and the two haemoglobins showed an alkaline Bohr effect larger than that of human haemoglobin A.     

A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.     

A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase.

One of the major components of rat liver mitochondria detected by gel electrophoresis in sodium dodecyl sulfate is a 165,000 molecular weight polypeptide that makes up 15 to 20% of the total mitochondrial protein. This component appears to be a single molecular species. Evidence is presented here for the identification of this protein with the polypeptide chain of a urea cycle enzyme, carbamoylphosphate synthetase I (EC 2.7.2.5). The 165,000 molecular weight polypeptide was solubilized from mitochondria with Triton X-100 and purified to 90% homogeneity by DEAE-cellulose chromatography. This component co-migrated with carbamyl phosphate synthetase activity when mitochondrial proteins were separated by gel filtration or sucrose gradient centifugation. The identification of the 165,000 molecular weight polypeptide with this activity was also supported by the presence or absence of this protein in a variety of rat tissue mitochondria, in liver and kidney mitochondria from various ureotelic and nonureotelic species, and in fetal rat liver mitochondria.     

Purification and properties of a DNA-dependent ATPase induced by bacteriophage T4.

A DNA-dependent ATPase formed after T4 phage infection is purified to apparent homogeneity. The molecular weight of the purified enzyme is 50 000 when determined by glycerol gradient centrifugation and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The enzyme at an earlier stage in purification (prior to DEAE-cellulose chromatography) exists as a complex with a molecular weight of 100000. However, molecular weight determinations by Sephadex gel chromatography give considerably decreased molecular weights for the complex and for the enzyme after DEAE-cellulose chromatography. The enzyme is stimulated to varying degrees by a variety of single-stranded polydeoxyribonucleotides or by single-stranded DNA, but no chemical change in the polynucleotide has been detected as a result of the enzyme action.     

Immunochemical study of the antigenic structure of bacteria of genus Bordetella. IV. Fractionation of B. pertussis extracts and study of the immunochemical and biological properties of the isolated fractions]

Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.     

Purification of the phosphofructokinase regulatory factors

In this paper, the existence and purification of two species of phosphofructokinase regulatory factor activity are reported. The purification procedure included liver homogenization and ultracentrifugation, a 93 degrees C heat step on the supernate, precipitation with ammonium sulfate, DEAE-cellulose column chromatography, and Sephadex G-75 (fine) chromatography. Two discrete regions of factor activity were eluted from the DEAE-cellulose column with a 0 to 0.5 M linear NaCl gradient. The lesser anionic fraction was not significantly retarded by DEAE-cellulose at pH 7.6, and was referred to as factor A. The more anionic form, factor B, eluted at about 0.2 M NaCl. The presence of two active fractions was confirmed by separation of factor activity (prior to DEAE-cellulose chromatography) into two discrete species by preparative isoelectric focusing on granulated gel. The isoelectric points were approximately 7.0 for factor B and 8.5 for factor A. Factor A and factor B exhibited quite different elution volumes, i.e., apparent molecular weights, when applied to a Sephadex G-75 column. Rechromatography on a Sephadex G-75 column was used for further purification and estimation of native molecular weight. The gel filtration method yielded a molecular weight of 13,800 +/- 1,800 for factor A. Factor A activity eluted as a symmetrical protein peak of constant specific activity, suggesting a homogeneous preparation. For factor B, the absorption at 280 nm and activity profile did not directly overlap. When the peak absorbance at 280 nm was considered, a molecular weight range of 39,000 +/- 4,000 was found, and on the basis of activity the molecular weight range was 36,000 +/- 4,000. After the final Sephadex G-75 chromatographic step, sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis of each SDS-treated factor preparation indicated that factor A, after visualization by silver staining, was homogeneous, with a subunit molecular weight of approximately 12,000. The factor B preparation consisted of two major polypeptides (11,000 and 18,000). The data appeared to support the conclusions that factor B was a dimer of the 18,000-Da subunit, and that the major contaminant was a tetramer of the 11,000-Da subunit.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.