Sephadex G-200 Gel Chromatography Research Articles

Direct Evidence on Incorporation of the Receptor into the Nuclei of Rat Ventral Prostate in the Form of the Complex with Dihydrotestosterone

Cytosol 9S receptor was prepared from the supernatant fluid at 105,000 X g of rat prostate homogenates by a Sephadex G-100 gel chromatography, and was labeled with 131I. The 131I-labeled 9S receptor retained the activity of forming a complex with 3H-dihydrotestosterone, similarly to the intact cytosol receptor, when examined by a sucrose density gradient centrifugation. When the 131I-labeled cytosol 9S receptor was incubated with isolated prostatic nuclei in the presence of 3H-dihydrotestosterone, it was found that the 131I-labeled receptor was directly incorporated into the nuclei in a form of the complex bound to 3H-dihydrotestosterone. 131I-Labeled receptor-3H-dihydrotestosterone complex which was incorporated into the nuclei was extracted with 0.5 M KCl solution, and the nuclear complex sedimented with a velocity of 5S. The incorporation of 131I-labeled receptor-3H-dihydrotestosterone complex into the nuclei increased along with the raised temperature of incubation, whereas association of the complex with the chromatin reached maximum at 35 degrees C and then decreased gradually beyond this temperature. In the time course study, either the incorporation of the complex into the nuclei or the association with the chromatin reached the maximal levels within 10 min and leveled off up to 60 min. On the other hand, 131I-labeled serum protein was far less efficiently incorporated into the nuclei than the radioiodinated 9S receptor, and association of the serum protein with the chromatin was limited to a very little extent. The 131I-labeled 9S receptor-dihydrotestosterone complex associated with the chromatin was found to be preferably distributed into the non-histone protein as well as the DNA itself of the chromatin. The radioactivity lost by dehistonization of the chromatin was almost negligible. Furthermore, the cytosol 9S receptor fraction bound to 3H-dihydrotestosterone was purified about 100 fold by the two consecutive column chromatographies. The partially purified receptor fraction which was labeled with radioiodine incorporated mostly into non-histone protein and DNA fractions.

Alpha-N-Benzoylarginine-2-naphthylamide Hydrolase (Cathepsin B1?) from Rat Skin. I. Preliminary Experiments with Skin Extract.

The cathepsin B1-like, alpha-N-benzoyl-DL-arginine-2-naphthylamide (BANA) hydrolyzing activity of rat skin extract was studied. The enzyme was extracted into low ionic strength buffers, and was activated by dithiothreitol, EDTA and KCN. Incubation of the extract at acidic pH resulted in a 3.6-fold increase in its BANA hydrolase activity. The presence of inhibitor(s) of BANA hydrolase in the skin extract was indicated by the non-linearity of the activity/enzyme concentration curve and by Sephadex G-100 gel chromatography, which also caused separation of several other skin proteases.

Ein Beitrag zur Herstellung von Antiserengegen losliche Blattproleine

Summary Antisera against extracts of soluble barley leaf proteins mainly contain antibodies to fraction I protein. A coarse elimination of fraction-I-protein by means of Sephadex G-200 gel chromatography or a 15 min temperature treatment at 55 °C permits the preparation of antisera with antibodies to 8—10 proteins as could be demonstrated by immuno-disc-electrophoresis.

Correlation between Mature and Immature Thymocytes

Low-dense mature thymocytes (MT) and high-dense immature thymocytes (IMT) were investigated. Immature thymocyte-potentiating factor (IMPF) was found in the supernatant from MT stimulated by Con A. On the other hand, suppressing activity was present in the supernatant from the unstimulated IMT. IMPF was different from Con A itself. Sephadex G-100 gel chromatography of IMPF revealed that the activity was recovered at the position between bovine serum albumin and ovalbumin.

Demonstration of a blocking factor in the plasma and spinal fluid of patients with subacute sclerosing panencephalitis. I. Partial characterization.

Conflicting reports on the immune responsiveness of patients with subacute sclerosing panencephalitis (SSPE) have been reported. This report shows that the leucocytes from four SSPE patients exhibited strong sensitivity to both measles and SSPE virus preparations as measured by the macrophage migration inhibition test, mixed lymphocyte virus infected cell culture test, and the lymphotoxin assay. Earlier suggestions that a factor may be operating to suppress cellular reactivity are confirmed by the demonstration that the response of lymphocytes from SSPE patients could be blocked by the addition of SSPE spinal fluid or plasma. It was determined that the blocking factor was stable at -20 degrees C, heat labile at 56 degrees C for 30 minutes, trypsin and neuraminadase sensitive, and had a mol wt greater than 150,000 as determined by Sephadex G-200 gel chromatography. The blocking factor appeared to be specific for SSPE virus and did not block the response of lymphocytes to nonspecific mitogenic agents and other viral and bacterial agents.

The Molecular Mechanism of the in Vitro 4 S to 5 S Transformation of the Uterine Estrogen Receptor

Abstract The rat uterine cytosol contains two estrogen-binding proteins (EBP) referred to as the and EBP. The 5 S EBP was produced by incubating the uterine cytosol-[3H]estradiol mixture at 28° for 30 min to promote the S to 5 S transformation as described by Jensen et al. ((1971) Biochem. Soc. Symp. 32, 133). These two EBP species were characterized in high ionic strength buffers using sucrose gradient centrifugation and Sephadex G-200 gel chromatography. With these analytical methods the sedimentation coefficient, molecular Stokes radii, and molecular weights were determined. In the presence of 0.4 m KCl at pH 7.4 the 4 S EBP has a sedimentation coefficient of 4.2 ± 0.04 S, a molecular Stokes radius of 44.0 ± 0.4 A, and a molecular weight of 76,200. While in the identical buffers the 5 S EBP has a sedimentation coefficient of 5.5 ± 0.02 S, a molecular Stokes radius of 58.5 ± 0.5 A, and a molecular weight of 132,700. In the presence of 0.4 m KCl and 3 m urea at pH 7.4 the 4 S EBP shows a decrease in its sedimentation to 3.6 ± 0.04 S, but an increase in its molecular Stokes radius to 53.8 ± 0.9 A. The estimated molecular weight is 79,900. The 5 S EBP has a sedimentation coefficient of 4.6 ± 0.9 S and a molecular Stokes radius of 70.6 ± 1.0 A. The molecular weight is 133,900. In addition, sucrose gradient centrifugation analyses, in the presence of 0.4 m KCl, 3 m urea at pH 7.4 showed that a moderate fraction (25 to 50%) of the 5 S EBP is dissociated to the 4 S EBP. In the presence of 0.4 m KCl at pH 6.8 the 4 S and 5 S EBPs sediment at 4.7 ± 0.04 and 5.6 ± 0.05 S, respectively. The 5 S EBP also shows a tendency to revert to the 4 S EBP. Consequently, it was observed that a combination of 0.4 m KCl and 3 m urea at pH 6.8 is extremely effective, in contrast to using each of these reagents alone, in dissociating the 5 S EBP to the 4 S EBP without causing a loss of [3H]estradiol binding. The dissociation process is first order with a half-life of approximately 5 hours. These results indicate that the in vitro S to 5 S transformation is an association of the 4 S EBP, having a molecular weight of ∼80,000, with a second component or subunit of ∼50,000 to form the 5 S EBP (∼130,000). Concurrent with the association process, the 4 S and 5 S EBPs are also capable of marked conformational changes that were discerned when the EBPs were compared to the protein standards under identical conditions. The protein standards used were myoglobin, trypsin, ovalbumin, serum albumin, alkaline phosphatase, aldolase, γ-globulin, and ferritin. The conformational changes of the EBPs were shown by their sedimentation coefficients and molecular Stokes radii varying in a reciprocating manner while their molecular weights remained constant.

Purification of an acidic deoxyribonuclease II from the primate spleen

1. 1. Acid DNase from monkey and baboon spleens was isolated and purified by extraction at pH 3–4, (NH 4 2SO 4 fractionation, heating at 50° C for I hour, SP-C 50 chromatography and Sephadex G-100 gel chromatography. The final yield was 22 per cent. 2. 2. The results of polyacrylamide disc gel electrophoresis, isoelectrofocusing and analytical ultracentrifugation indicated that the preparations were pure and homogeneous. 3. 3. The optimum pH for the hydrolysis of highly polymerized DNA by the two acid DNases is 5.2. 4. 4. The two enzymes had essentially the same sedimentation coefficients (s 20 0, w, 2.5 × 10 1 ̄ 3 second) , diffusion coefficients ( D 20,w, 8 × 10 −7 cm 2. second −1), partial specific volumes ( v, 0.73 cm. 3 g. −1) , molecular weights (mol. wt. approximately 38,000), and isoelectric points (pI, 6.2).

Protein catabolism. II. Identification of neutral and acidic proteolytic enzymes in Aspergillus niger

Two proteinases of Aspergillus niger, one of pH optimum 3·4, the other of pH optimum 7·4, were purified 840- and 740-fold by (NH 4) 2SO 4 precipitation Sephadex G-100, G-150 and G-200 gel chromatography. Both proteinases were assayed using [ 3H]acetyl hemoglobin as substrate. The purified pH 3·4 proteinase had two bands on sodium dodecylsulfate polyacrylamide gel electrophoresis, while the purified pHH 7·4 proteinase gave one band when stained for protein; none of the purified material stained for glycoprotein. The purified pH 3·4 proteinase had molecular weights of 49 000 and 56 000, pI of 4.1 and 4.5 and a K m of 1·10 −5 M. This acid proteinase showed no cofactor requirements, was inhibited 51% by o.1 M HgCl 2, and was relatively inactive against synthetic substrates of low molecular weight. Analysis of the products of digestion of the [ 3H]acetyl hemoglobin after 1 h digestion with this enzyme showed that 71% of the product consisted of peptides of molecular weights less than 4000. The purified pH 7·4 proteinase had a molecular weight of 68 000, pI of 6.2, and a K m of 5·10 −5 M. This neutral proteinase showed no cofactor requirements, was severely inhibited by HgCl 2 and diisopropylphosphofluoridate and was also relatively inactive against low molecular weight synthetic substrates. Analysis of the products of digestion with this neutral proteinase showed that 66% of the product consisted of peptides of molecular weight less than 4000.

BIOSYNTHESIS OF HUMAN CHORIONIC GONADOTROPHIN IN VITRO: INCORPORATION OF [14C]l-LEUCINE

SUMMARY Fresh human placental tissue from first trimester pregnancies was incubated with [14C]l-leucine and the incorporation of the labelled amino acid into placental proteins was studied in both the soluble and the particulate fractions. Free [14C]l-leucine was completely separated from labelled proteins by exhaustive dialysis. This was confirmed by Sephadex G-100 gel chromatography. In preliminary experiments, the total protein fraction from Sephadex was submitted to polyacrylamide gel electrophoresis; columns divided into ten sections were checked, fraction by fraction, for incorporated radioactivity and human chorionic gonadotrophin (HCG) potency, using a radioimmunoassay method. In all instances, the highest HCG activity corresponded to the highest incorporation of radioactivity. In subsequent experiments only those radioactive proteins were used which had the highest activity after chromatography. This peak was further divided into two parts which were submitted to polyacrylamide gel electrophoresis. Using the ascending limb of the peak which contained about 70% of the total HCG activity, there was complete agreement between the distribution of radioactivity and that of HCG activity, whereas the bulk of HCG radioimmunological activity in the descending limb could be dissociated from radioactive proteins. After absorption of the HCG-like protein with specific anti-HCG serum, HCG activity was completely neutralized and more than 95% of the corresponding radioactivity disappeared. Additional evidence for the presence of an HCG-like biosynthesized protein was obtained after DEAE-cellulose ion exchange chromatography of the products of the incubation. Here again, the HCG potency of the radioactive protein with the highest activity was completely neutralized after absorption with a specific antiserum: in this case, however, only some 60% of the total radioactivity present disappeared after absorption. The results indicate that human placental tissue can synthesize from l-leucine proteins possessing radioimmunological HCG activity.

β-Cyclopiazonate oxidocyclase from Penicillium cyclopium. I. Assay methods, isolation and purification

1. 1. Spectrophotometric and colorimetric assay methods for the dehydrogenation of β-cyclopiazonic acid and the conversion of β- into α-cyclopiazonic acid, both catalysed by β-cyclopiazonate oxidocyclase, were developed. Paper chromatography, ultraviolet spectrophotometry and mass spectrometry were also used in identifying the product, α-cyclopiazonic acid, formed in the above conversion. 2. 2. Five isoenzymes of β-cyclopiazonate oxidocyclase were isolated and purified from the mycelium of Penicillium cyclopium Westling by homogenization and extraction, differential centrifugation, (NH 4) 2SO 4 fractionation, DEAE-cellulose chromatography, Sephadex G-100 gel chromatography and CM-Sephadex C 50 chromatography. The final yield was 64% and the average final enrichment for the five isoenzymes 133. 3. 3. The results of microzone electrophoresis, polyacrylamide gel electrophoresis and analytical ultracentrifugation indicated pure and homogeneous preparations of all five isoenzymes. 4. 4. The optimum pH for the dehydrogenation reaction and the conversion of β- into α-cyclopiazonic acid was found to be 6.8. 5. 5. The five isoenzymes had essentially the same sedimentation coefficients ( s o 20, w approx. 4.0·10 −13 sec); diffusion coefficients ( D 20, w approx. 7.3·10 −7 cm 2·sec −1) partial specific volumes ( ν, 0.73 cm 3· g −1 ) and molecular weights (mol. wt. approx. 50 000). 6. 6. The isoenzymes showed a similar specific activity for both the dehydrogenation and the conversion of β- into α-cyclopiazonic acid and occurred in a constant relative ratio.

Identification of one of the L-serine dehydratase isoenzymes from rat liver as L-homoserine dehydratase

The higher molecular weight L-serine dehydratase was purified from rat liver and fractionated into two active components by Sephadex G-200 gel chromatography. On the basis of molecular weight, ultracentrifugal pattern and substrates specificity of the major component, it was concluded that this component is identical to L-homoserine dehydratase. Some misinter-pretations which have been derived from the work on L-serine dehydratase in animal tissues are pointed out.

The glycogen phosphorylase of Tetrahymena pyriformis: I. Purification and characterization

Glycogen phosphorylase (α-1,4-glucan; orthophosphate glucosytransferase, EC 2.4.1.1) was purified 130-fold from acetone powder prepared from Tetrahymena pyriformis. The most purified preparation was about 40% pure as judged by polyacrylamide disc gel electrophoresis and had a specific activity of 4.1 μmoles P i/min/mg protein. The enzyme also catalysed the phosphorolysis of glycogen and is thus a true glycogen phosphorylase. The molecular weight of the enzyme was about 200,000 as determined by sucrose density gradient centrifugation, but two peaks, corresponding to molecular weights of 100,000 and 200,000 were observed on Sephadex G-200 gel chromatography. The enzyme was slowly inactivated at temperatures up to 50 ° but a sharp increase in the rate of inactivation occurred above 50 °. AMP, but not glucose-1-phosphate or glycogen, partially protected the enzyme against thermal inactivation. AMP and structurally related nucleoside monophosphates neither stimulated nor inhibited the activity of the enzyme.

Verteilung der l-alanyl-peptid-hydrolase aktivitat im menschlichen serum und in organ-homogenate mit hilfe der sephadex g-200 gel-chromatographie

The distribution of ANAase activity in human organ homogenates mixed with normal human serum and in human hepatitis sera respectively, was investigated by Sephadex G-200 gel chromatography. In homogenates of the investigated organs (kidney, duodenum, jejunum, pancreas and liver) and in hepatitis sera two peaks of ANAase activity were found. One of them corresponds approximately to molecular weight of 150000 and the other of 450000–500000. On all ANAase activity diagrams small additional irregularly occurring peaks were present. In jejunum homogenate a relatively high ANAase activity peak was found, corresponding to 45000 M.W. and in duodenal homogenate another one, which corresponded to 280000 M.W. The highest ANAase activity was found in kidney homogenate (8.95 IU/g protein) and the lowest in liver homogenate (2.43 IU/g protein). In hepatitis serum the ANAase activity was 1.63 IU/g protein and in normal serum 0.44 IU/g protein. The factors possibly influencing the distribution of ANAase activity after Sephadex G-200 gel chromatography were briefly discussed.

Nonidentiy of some simian virus 40-induced enzymes with tumor antigen.

The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the time of infection with 1-beta-d-arabinofuranosylcytosine (ara-C), and from SV40-transformed cells. Upon partial purification, the T antigen exhibited the following properties: it was tightly adsorbed by calcium phosphate gel, it was precipitated by acetic acid at pH 5 or by ammonium sulfate at about 20 to 32% saturation, and it had a molecular weight greater than 250,000, as estimated by Sephadex G-200 gel chromatography. In contrast, deoxycytidylate (dCMP) deaminase, thymidylate (dTMP) kinase, and thymidine (dT) kinase were less strongly bound to calcium phosphate and were not precipitated at pH 5; these enzymes also had much lower molecular weights than the T antigen, as did dihydrofolic (FH(2)) reductase. Furthermore, higher ammonium sulfate concentrations were required to precipitate dCMP deaminase, dTMP kinase, and FH(2) reductase activities than to precipitate the T antigen. Another difference was that the T antigen was not stabilized, but dCMP deaminase, dTMP kinase, and dT kinase, were stabilized, respectively, by dCTP, dTMP, and dT or dTTP. Deoxyribonucleic acid (DNA) polymerase activity resembled the T antigen in adsorption to calcium phosphate, in precipitation by ammonium sulfate or at pH 5, and in the rate of inactivation when incubated at 38 C. However, the polymerase activity could be partly separated from the T antigen by Sephadex G-200 gel chromatography. The cell fraction containing partially purified T antigen also contained a soluble complement-fixing antigen (presumably a subunit of the viral capsid) which reacted with hyperimmune monkey sera. The latter antigen was present in very low titers or absent from cell extracts prepared from SV40-infected monkey kidney cell cultures which had been treated with ara-C at the time of infection, or from SV40-transformed mouse kidney (mKS) or hamster tumor (H-50) cells. The T antigen, however, was present in usual amounts in SV40-transformed cells or ara-C treated, infected cells.