Purification and some properties of a deoxyribonucleic acid endonuclease endogenous to rat liver chromatin

A deoxyribonucleic acid (DNA) endonucleolytic activity has been purified from a 0.3 M KCl extract of rat liver chromatin by a combination of selective precipitation and ion-exchange and gel filtration chromatography. The purified protein has a molecular weight of 35 000 as determined by Sephadex G-200 gel filtration and sodium dodecyl sulfate-acrylamide gel electrophoresis. The nuclease activity is stimulated by the addition of Mg2+ and thus may represent the Mg2+-activated DNase endogenous to chromatin. The purified enzyme has the ability to make both single-strand nicks and double-strand cuts in DNA.     

Heterogeneity of big-big hPRL in hyperprolactinemia

Sera from a patient with macroprolactinoma (case 1) and from a hyperprolactinemic woman with regular menstruation (case 2) were analyzed for prolactin activity by gel filtration using Sephadex G-100, Sephadex G-200 and TSK G3000SW columns. The chromatographic profile by Sephadex G-100 showed that the percentage of immunoreactive big-big hPRL was 10.7% in case 1 and 64.1% in case 2. On Sephadex G-200 and TSK G3000SW columns, the molecular weight of big-big hPRL was estimated to be more than 500,000 daltons (big-big1 hPRL) in case 1 and approximately 250,000-300,000 daltons (big-big2 hPRL) in case 2. Big-big1 hPRL in case 1 was converted to big and little hPRLs when the serum was treated with 2-mercaptoethanol (2-ME), but part of the big-big2 hPRL in case 2 was converted to a larger molecule. Radioactive big-big hPRL generated by mixing labeled hPRL with the serum from case 1 was eluted with the void volume on Sephadex G-100 column and was not converted to the other molecular forms after 2-ME treatment. There were two radioactive big-big hPRL on TSK G3000SW column and these estimated molecular weights were more than 300,000 daltons. The data demonstrated the existence of at least two forms of big-big hPRL in the serum and indicated that radioactive big-big hPRL may be different from these hPRLs in the serum.     

Distribution and Properties of Chromatin-Associated Nucleoside Triphosphate Diphosphatase Purified by a New Method

Chromatin-associated nucleoside triphosphate diphosphatase (NTDPase)activity was detected in Alaska pea plant only at the germinationstage in a study of the plant's development over 5 month fromthe dormant to fruiting stages. This enzyme activity was alsodetected in seedlings of several dicotyledonous plants includingsoybean, Usui pea, cowpea and cucumber, but almost none wasfound in monocotyledonous corn and rice plants. When the chromatin of pea epicotyl was fractionated into template-activeand -inactive forms by DNase II digestion, most of the NTDPaseactivity was found in the active chromatin. A new method to rapidly isolate and purify NTDPase from peaepicotyl chromatin was developed in which NTDPase was elutedwith a linear gradient of NaCl on a column packed with cellulosepowder and chromatin. DNA remained on the column and the elutedNTDPase was purified further by chromatography using trimethylamino-2-hydroxypropylcellulose (TMAP-cellulose) and a Sephadex G-100 column, whichgive chromatin 18-fold purer than the starting sample. This purified NTDPase was adsorbed by DNA-cellulose, which isfurther evidence that NTDPase is a kind of non-histone proteinassociated with DNA. Several cations including Ca^2$, Mg^2$, Mn^2$and Zn^2$ stimulated the enzyme activity, with the maximal eightfoldactivation by Ca^2$. NTDPase activity was clearly inhibited byKCN and pyrophosphate, but not by SH-blocking inhibitors andvarious metal chelators at 1 mM.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.     

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.     

Hepatoma-associated non-histone proteins are phosphoproteins preferentially localized in nuclear matrix

1. High molecular weight non-histone proteins (NHP) were isolated from Morris hepatoma 7777 by Sephadex G-100, S-200 chromatography. 2. Specific polyclonal antibodies were raised against these NHP in rabbits. These antibodies recognized specific NHP components present in Morris hepatoma 7777 and 8994, but not in normal rat liver. Hepatoma-associated antigens are phosphoproteins. 3. Immunologically specific NHP of Morris hepatoma are intensively concentrated in nuclear matrix fraction.     

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.     

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 μg/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.     

Starch phosphorylase from tapioca leaves: Absence of pyridoxal phosphate

Starch phosphorylase from tapioca leaves has been purified to homogeneity, using the technique of ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, filtration through Sephadex G-100 and Sephadex G-200, and DEAE-Sephadex chromatography. The enzyme has a molecular weight of 450,000, as determined by gel filtration through Sephadex G-200 and contains 22 sulfhydryl groups per mole of the enzyme protein. Several types of evidence indicate the absence of pyridoxal 5′-phosphate as a prosthetic group of the enzyme. The kinetic data show a sequential type of the reaction mechanism. The enzyme activity is inhibited by tyrosine (K_i = 2.15 mm).     

The thermodynamics of interaction between Sephadex and penetrating solutes

1. We have measured the partition coefficients of bovine serum albumin with Sephadex grades G-100, G-150 and G-200, and of a dextran ([unk]_n 19700) and a polyethylene glycol ([unk]_n 8000) with Sephadex G-200. We have also measured the effects of these solutes on the inner volumes of the grades of Sephadex. 2. The results can be described with fair consistency by means of a simple thermodynamic treatment that makes use of the virial coefficients of Sephadex and of the solute, and of a coefficient that expresses their interaction. This coefficient is related to the `exclusion volume' of Sephadex for the solutes. 3. The Sephadex G-200–polyethylene glycol system shows anomalies of behaviour that are ascribed to the occurrence of `incompatible' phase separation within the Sephadex beads.     

Purification of angiotensin I-converting enzyme from human lung.

Angiotensin I-converting enzyme (peptidyl dipeptide hydrolase, EC 3.4.15.1) was solubilized from the membrane fraction of human lung using trypsin treatment and purfied using columns of DE 52-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 9.5 units/mg protein for Hippuryl-His-Leu-OH and 0.665 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment (1 mg/200 mg protein) for 2 h could be divided into three components: (i) an enzyme of molecular weight 290 000 (peak I), (ii) an enzyme of molecular weight 180 000 (peak II) and (iii) an enzyme of molecular weight 98 000 (peak III), by columns of DE 52-cellulose and Sephadex G-200. Km values of peak I, II and III fraction for Hippuryl-His-Leu-OH were identical at 1.1 mM. pH optimum of the enzyme was 8.3 for Hippuryl-His-Leu-OH.     

Partial purification and some properties of gamma-glutamyl transpeptidase from human bile.

1. Gamma-Glutamyl transpepetidase ((5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2) from human bile has been partially purified using protamine sulphate treatment, DEAE-cellulose chromatography and Sephadex G-200 filtration. The procedure resulted in 150-fold increase in specific acitivity with a 37% yield. 2. The partially purified enzyme showed a single zone of enzyme activity by polyacrylamide gel electrophoresis and eluted in the inner volume of Sephadex G-200. 3. The enzyme had a pH optimum of 8.1 and Km of 1.52 mM using gamma-glutamyl p-nitroanilide as substrate. 4. The effects of cations and different gamma-glutamyl acceptors on the activity of the enzyme are reported. 5. As bile gamma-glutamyl transpeptidase appears to be soluble in the absence of detergents, it is suggested that bile may prove to be a useful source for further studies of the kinetic properties and physiological role of human gamma-glutamyl transpeptidase.     

Solubilization of angiotensin I-coverting enzyme from rabbit lung using trypsin treatment.

The solubilization of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was carried out using trypsin treatment. A good recovery of 76% was obtained. The enzyme from solubilized fraction was purified using colums of Sephadex G-200, hydroxyapatite and DEAE-cellulose. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 24.3 units/mg protein for hippurylhistidylleucyl hydroxide and 0.182 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment for 5 h could be divided into two components: (i) an enzyme of molecular weight 300 000 (peak II) and (ii) an enzyme of molecular weight 145 000 (peak III), by Sephadex G-200 gel filtration. The molecular weight of the denatured enzyme was found to be 155 000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Km values of peak II and peak III fraction for Hippuryl-His Leu-OH were 2.6 mM.     

Isolation and characterization of a sulfated glycoprotein from rabbit uterus.

Crude glycoproteins were extracted with 0.15 M NaCl from the pooled endometrial scrapings of rabbit uteri after treatment with estrogen. The crude glycoproteins were fractionated with ammonium sulfate, followed by DEAE-cellulose column chromatography, treatment with CM-Sephadex C-25 and gel filtration on Sephadex G-200. Subsequently, purification of an acidic glycoprotein was carried out by gel filtration on Sephadex G-200 and then Sepharose 4B. The results of electrophoresis and enzymatic digestion, together with analytical data and the infrared spectrum indicated that the acidic glycoprotein was a sulfated glycoprotein.     

Properties of delta5-3beta-hydroxysteroid oxidoreductase isolated from Streptomyces griseocarneus.

Delta5-3beta-hydroxysteroid oxidoreductase was extracted in magnesium-containing Tris buffer from sonicated Streptomyces griseocarneus cells. The enzyme was partially purified (150 X) by ion exchange chromatography and gel filtration following (NH4)2SO4 fractionation. Upon gel filtration on Sephadex G-75 to G-200, the greatest part of the activity gave a peak in the fractionation range. The enzyme obtained from the gel yielded small enzyme molecules on repeated chromatography. A molecular weight of 32 to 36 000 was calculated for the activity appearing in the fractionation range of Sephadex G-75 to G-200. The enzyme is highly specific for the irreversible oxidation of the 3beta-hydroxyl group in steroids with a trans-anellated A : B ring system with either C5 or C6 double bond. Delta5-3-ketosteroids are converted into delta5-3-ketosteroids at a high rate, but the isomerase activity cannot be separated from the oxidoreductase activity either by chromatography or by selective heat inactivation. NAD, NADP, FMN or FAD did not influence the activity, but the enzyme is inactive in the absence of molecular oxygen.     

Density-Gradient and Chromatographic Fraction of Leptospiral Lipase

Fractionation of leptospiral lipase by CsCl density gradients and G-200 Sephadex chromatography yielded five active protein peaks. Two were obtained from the density gradients and three from G-200 Sephadex columns. Esterase activity of these fractions was demonstrated by electrophoretic examination. Several protein bands were visible when disc electrophoresis was performed on the respective fractions. Lipolytic and esterolytic activities were both present, and the overlapping of these activities was discussed.     

The inhibition of a chromatin-bound proteolytic activity

A chromatin-bound proteolytic activity found in rabbit thymus nuclear lysates has been studied. Incubation of washed chromatin fibers led to degradation of histone H1 resulting in products which ran on sodium dodecyl sulfate-gels slightly ahead of the three native H1 fractions. The activity has also been demonstrated in a chromatin preparation purified by chromatography on Sephadex G-200. Of the five histones, H1 appeared to be the most readily degraded although with extended incubation times the core histones were also affected. The proteolytic activity was not inhibited by Na₂SO₃, PMSF, STI, pepstatin, or EDTA. Effective inhibitors included leupeptin, tosyllysine chloromethyl ketone, and tosylaminophenylethylchloromethyl ketone.     

Arginine metabolism in Saccharomyces cerevisiae. Some general properties of yeast arginase

Cells of Saccharomyces cerevisiae ATCC 9763 were cultured aerobicallyat 30?C in defined media containing arginine as a sole sourceof nitrogen. After 5 hr of growth arginase (L-arginine ureohydrolaseEC 3.5.3.1 [EC] ) was extracted by sonication and partially purifiedby gel filtration. The specific enzyme activity was increasedthirty times as a result of Sephadex G-200 treatment. The geltreated enzyme was stable for at least 3 days when stored at2?C. Enzyme activity was maximal at pH 9.2 and was increasedby preincubation with divalent metallic ions. The apparent Michaelisconstant for arginine was 12.5 mM in glycinate buffer and 250mil in phosphate buffer. The enzyme was strongly inhibited byL-ornithine. The kinetics of this inhibition revealed that ornithinecompeted with arginine for the active site. The molecular weightof yeast arginase was estimated to be 120000?10000 by determinationof elution volumes from Sephadex G-200. The general propertiesof the yeast enzyme are discussed in relation to (a) arginasesfrom other sources and (b) regulation of the Krebs-Henseleitcycle in Saccharomyces. (Received January 27, 1973; )