scholarly journals Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues

1992 ◽  
Vol 288 (1) ◽  
pp. 233-240 ◽  
Author(s):  
M T Tuck
Keyword(s):  
Rat Liver ◽  
Rat Kidney ◽  
Cell Activity ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Methyltransferase Activity ◽  
Nuclear Extracts ◽  
Normal Rat ◽  
Minor Form

Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5′ cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity.

scholarly journals THE ENZYMATIC HYDROLYSIS OF AMINO ACID β-NAPHTHYLAMIDES II. PARTIAL PURIFICATION AND PROPERTIES OF A PARTICLE-BOUND COBALT-ACTIVATED RAT KIDNEY AMINOPEPTIDASE

1966 ◽  
Vol 14 (5) ◽  
pp. 401-413 ◽  
Author(s):  
K. FELGENHAUER ◽  
G. G. GLENNER
Keyword(s):  
Amino Acid ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Disc Electrophoresis ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Solvent Fractionation ◽  
Biochemical Systems ◽  
Hydrolysis Of

In order to determine the relationship and characteristics of the soluble and tissue-bound (so-called "lyo" and "desmo") components of enzymes in histochemical and biochemical systems, a biochemical investigation of the hydrolysis of l-leucyl β-naphthylamide in rat kidney was undertaken. By ultracentrifugation, autolysis, salt and solvent fractionation procedures and DEAE-cellulose column chromatography primary soluble and tissuue-bound enzymes were separated and partially purified. The soluble enzyme was found to be inhibited by p-chloromercuribenzoate and reactivated by cysteine. The tissue-bound enzyme was found to be inhibited by o-phenanthroline and reactivated by Co++, and hydrolyzed peptides and amino acid β-naphthylamides at rates differing markedly from those of the soluble enzyme and commercial leucine aminopeptidase. Disc electrophoresis of the tissue-bound enzyme preparation demonstrated two protein bands, both revealing enzymic activity with almost identical hydrolytic characteristics, i.e., isozymes. This evidence established the identity of the tissue-bound Co++-activated enzyme with that found histochemically in the brush border of the proximal tubuli, and indicated its functional capacity as that of an aminopolypeptidase. It was also concluded that in the system investigated the "lyo" and "desmo" components represented, in reality, distinct enzymes with unique characteristics.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

1988 ◽  
Vol 55 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Efstathios Alichanidis
Keyword(s):  
Aeromonas Hydrophila ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Significant Activity ◽  
Metal Chelators ◽  
Purification And Characterization ◽  
Tris Maleate ◽  
Ph Range

SummaryAn extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatogrpahy on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43000 and contained 2 g atom Ca/mol. It was active over a pH range 4·8–9·5 and had optimum activity on casein at pH 7·0 with Km = 0·17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 °C; above 50 °C activity declined rapidly, but significant activity persisted at 4 °C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

scholarly journals Extraction of an Erythropoietin-Producing Factor from a Particulate Fraction of Rat Kidney

Blood ◽  
1966 ◽  
Vol 28 (3) ◽  
pp. 330-343 ◽  
Author(s):  
JOSEPH F. CONTRERA ◽  
ALBERT S. GORDON ◽  
ARTHUR H. WEINTRAUB
Keyword(s):  
Rat Kidney ◽  
Deae Cellulose ◽  
Mitochondrial Fraction ◽  
Normal Serum ◽  
The Other ◽  
Step Method ◽  
Renal Vasculature ◽  
Rat Serum ◽  
Normal Rat ◽  
Strong Resemblance

Abstract 1. A two-step method for the extraction of erythropoietin from hypoxic kidneys has been developed which allows residual plasma erythropoietin in renal vasculature to be separated from that of intracellular origin. 2. Renal extracts have been purified by DEAE cellulose chromatography and found to contain 2 major erythropoietically active fractions. One bears strong resemblance to plasma erythropoietin. The other component is unique in that it has practically no erythropoietic-stimulating activity unless previously incubated with normal rat serum. This activation phenomenon is used to identify this kidney component as the renal erythropoietic factor (REF). The REF has the capacity to produce erythropoietin or become erythropoietically active when incubated with normal rat serum. 3. Differential centrifugation technics revealed that the REF is confined to particles present in the light mitochondrial fraction of kidney. 4. Extracts of the light mitochondrial fraction of kidneys from normal rats produced significant amounts of erythropoietin when incubated with normal serum. The quantity found, however, was less than that evoked by similar extracts of kidneys from hypoxic rats. 5. The product of the incubation extracts of the renal light mitochondrial fraction with normal rat serum showed the same log dose/response regression as sheep plasma erythropoietin standard. 6. It is hypothesized that either (a) the REF is a precursor of erythropoietin which must be complexed with a carrier present in normal serum in order to become physiologically active, or (b) the renal factor is an enzyme which produces erythropoietin by its action on a particular serum protein.

INTRACELLULAR DISTRIBUTION OF DESOXYRIBONUCLEO-DEPOLYMERASE IN NORMAL RAT LIVER, LIVER TUMOR, AND LIVER OF ANIMALS FED p-DIMETHYLAMINOAZOBENZENE

1954 ◽  
Vol 32 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Gaston de Lamirande ◽  
Claude Allard ◽  
Antonio Cantero
Keyword(s):  
Rat Liver ◽  
Liver Tumor ◽  
Cell Activity ◽  
Mitotic Rate ◽  
Intracellular Distribution ◽  
Nuclear Fraction ◽  
Normal Rat ◽  
High Level ◽  
Soluble Material

The intracellular distribution of desoxyribonucleodepolymerase (DNase) has been investigated in the liver of animals fed p-dimethylaminoazobenzene (DAB), in liver freed from tumor, and in DAB induced tumor. The method is based on the determination of acid soluble material containing phosphorus, liberated by the action of the enzyme upon highly polymerized DNA. Results indicated that the nuclear DNase particularly accounts for a very low percentage of the whole cell activity in normal rat liver, whereas in nuclei of liver of DAB fed rats and of tumor the activity is increased to a high level. These facts suggest a possible correlation between the activity of DNase in the nuclear fraction and the mitotic rate of the tissue.

scholarly journals Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells

1983 ◽  
Vol 213 (2) ◽  
pp. 379-386 ◽  
Author(s):  
G J Strewler ◽  
M A Danello ◽  
V C Manganiello ◽  
M Vaughan
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Gmp ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Hepatoma Cells ◽  
Partial Hydrolysis ◽  
Kinetic Behaviour ◽  
Lysosomal Proteinase ◽  
Hydrolysis Of

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.

scholarly journals Identification and properties of renal mineralocorticoid receptors in relation to glucocorticoid binders in rat liver and kidney

1976 ◽  
Vol 154 (3) ◽  
pp. 567-575 ◽  
Author(s):  
M K Agarwal
Keyword(s):  
Molecular Weight ◽  
Blood Serum ◽  
High Molecular Weight ◽  
Rat Liver ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Competitive Binding ◽  
Mineralocorticoid Receptors ◽  
Liver And Kidney

The binding of the natural mineralocorticoid aldosterone and the glucocorticoid corticosterone to macromolecules in rat liver and kidney cytoplasmic fractions was compared by various chromatographic procedures. Equilibration of kidney cytosol with 10nM-aldosterone, either alone or in the presence of a competing steroid, was ideal for ionexchange chromatography of DEAE-cellulose DE-52, and revealed the presence of four sorts of binding components. One of these, eluted in the 0.001M-phosphate pre-wash, and another, less abundant, forming a peak at 0.006M-phosphate, did not bind corticosterone at equimolar concentrations, and appear to constitute the mineralocorticoid-specific ‘MR‘ receptor in rat kidney. They could not be detected in the liver. Radioactivity eluted in the 0.02 and 0.06M-phosphate regions on DEAE-cellulose DE-52 appears to be due to [3H]aldosterone binding to glucocorticoid-specific ‘GR’ receptors and to transcortin respectively, since labelling was greater with corticosterone even at 10 nM than with the mineralocorticoid at 100nM and since [14C]corticosterone bound to blood serum transcortin was always co-chromatographed in the 0.06M-phosphate region. These two components appear to be identical with those in the liver and could be labelled maximally only by 100nM-corticosterone. The separation between specific mineralo- and glucocorticoid-binding species was less clear when chromatography was attempted on DEAE-Sephadex A-50 columns, possibly because of disaggregation into subunits in the presence of the high KC1 concentrations required for elution. Competitive binding followed by filtration through Sephadex G-200 gel indicated that cellular MR binders, unlike GR receptors, exist mostly as high-molecular-weight aggregates, although both appear to exhibit a comparable monomeric molecular weight of approx. 67000.

scholarly journals Some properties of galactokinase in developing rat liver

1968 ◽  
Vol 108 (2) ◽  
pp. 169-175 ◽  
Author(s):  
D G Walker ◽  
H. H. Khan
Keyword(s):  
Kinetic Parameters ◽  
Rat Liver ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Dietary Factors ◽  
Activity Profile ◽  
Adult Rats ◽  
Stage Of Development ◽  
Developing Rat

1. The nature of the galactokinase present in the livers of foetal, newborn and adult rats was examined by the application of several separation procedures and by measurement of a range of kinetic parameters. 2. No evidence of enzyme heterogeneity at any stage of development was found during gel filtration on Sephadex G-100, column chromatography on DEAE-cellulose or a variety of electrophoretic procedures. 3. The Km values, inhibition characteristics and other kinetic parameters appear to remain constant during development. 4. Rat liver galactokinase activity does not adapt to dietary changes in either the adult or the newborn rat; hence it is unlikely that the presence of galactose in milk controls the enzymic activity profile during development. 5. On the present evidence it is concluded that only one form of galactokinase is present in rat liver and that the enzymic activity is controlled by non-dietary factors.

INTRACELLULAR DISTRIBUTION OF DESOXYRIBONUCLEO-DEPOLYMERASE IN NORMAL RAT LIVER, LIVER TUMOR, AND LIVER OF ANIMALS FED p-DIMETHYLAMINOAZOBENZENE

1954 ◽  
Vol 32 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Gaston de Lamirande ◽  
Claude Allard ◽  
Antonio Cantero
Keyword(s):  
Rat Liver ◽  
Liver Tumor ◽  
Cell Activity ◽  
Mitotic Rate ◽  
Intracellular Distribution ◽  
Nuclear Fraction ◽  
Normal Rat ◽  
High Level ◽  
Soluble Material

The intracellular distribution of desoxyribonucleodepolymerase (DNase) has been investigated in the liver of animals fed p-dimethylaminoazobenzene (DAB), in liver freed from tumor, and in DAB induced tumor. The method is based on the determination of acid soluble material containing phosphorus, liberated by the action of the enzyme upon highly polymerized DNA. Results indicated that the nuclear DNase particularly accounts for a very low percentage of the whole cell activity in normal rat liver, whereas in nuclei of liver of DAB fed rats and of tumor the activity is increased to a high level. These facts suggest a possible correlation between the activity of DNase in the nuclear fraction and the mitotic rate of the tissue.

scholarly journals Purification and characterization of a lysosomal form and a variant form of β-glucuronidase from the rat basophil leukaemia tumour

1981 ◽  
Vol 193 (3) ◽  
pp. 663-670 ◽  
Author(s):  
L B Schwartz ◽  
K F Austen
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Rat Tissues ◽  
Variant Form ◽  
Polyacrylamide Gels ◽  
Purification And Characterization ◽  
Normal Rat ◽  
Single Subunit

Two isoenzyme of beta-glucuronidase from a rat basophil leukaemia tumour were co-purified 4067-fold by (NH4)2SO4 precipitation and sequential chromatography on concanavalin A–Sepharose, Sephadex G-200, DEAE-cellulose, CM-cellulose and phosphocellulose. The purity of the mixture was established by the coincidence of the peaks of enzyme activity and protein at a molecular weight of 300 000 on Bio-Gel P-300, the presence of only two protein bands, both of them enzymically active, in polyacrylamide gels after electrophoresis under non-denaturing conditions, and the presence of a single subunit species, of mol.wt. 75 000, after electrophoresis in polyacrylamide gels under a denaturing conditioning. The major isoenzyme co-migrated with the L form from rat liver during electrophoresis in alkaline polyacrylamide gels, whereas the minor isoenzyme migrated more rapidly than either the lysosomal form or the rat liver microsomal form and was designated the tumour (T) isoenzyme. A mixture of the purified isoenzymes from two preparations had an average specific activity of 1389 units/mg for phenolphthalein beta-D-glycopyranosiduronic acid. The L and T isoenzymes, which had pI5.9 and 5.7 respectively, could be obtained free of cross-contamination by isoelectric focusing and had similar specific activities. Although the T isoenzyme could be a catabolic product of the M or the L form, it could also be a unique tumour product, because it was not detected in extracts of normal rat tissues.

Characterization and Partial Purification of Heterodisperse Polysomal RNAs in Normal and Neoplastic Cells

1972 ◽  
Vol 58 (2) ◽  
pp. 71-94
Author(s):  
Ada Sacchi ◽  
Gianni Chinali ◽  
Susetta Pons ◽  
Michela Galdieri ◽  
Piero Cammarano
Keyword(s):  
Size Distribution ◽  
Density Gradient ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Partial Purification ◽  
Messenger Rnas ◽  
Alkaline Ph ◽  
Sedimentation Profile ◽  
Molecular Weights ◽  
Phenol Extraction

The size distribution of cytoplasmic messenger RNAs (m-RNA) has been studied in rat liver and in monodifferentiated cells (mouse reticulocytes and myelomas). It has been found that the RNA which exhibits a « rapid turnover » and a polydisperse profile of radioactivity is refractory to phenol extraction. This property has been exploited to selectively isolate m–RNA from the phenol residue by means of an extraction at an alkaline pH. The sucrose density gradient profiles of m–RNA isolated from monodifferentiated cells show monodisperse peaks having the sedimentation coefficients expected on the basis of the molecular weights of monocistronic messages for α and β chains of hemoglobin (reticulocytes) and L and H chains of immunoglobulin (myelomas). The sedimentation profile of cytoplasmic m–RNA associated with rat liver polysomes shows a much broader distribution, with sedimentation coefficients ranging from 8 S to 28 S.

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