Ox Brain Aminopeptidases, Further Purification of Three Bovine Enzymes

1971 ◽  
Vol 49 (6) ◽  
pp. 676-685 ◽  
Author(s):  
R. E. Sobel ◽  
A. S. Brecher
Keyword(s):  
Ammonium Sulfate ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Heat Stability ◽  
Sucrose Density Gradient ◽  
Bovine Brain ◽  
Supernatant Fraction ◽  
Differential Heat ◽  
Sucrose Density Gradient Centrifugation ◽  
Brain Extracts

Four peptidases capable of acting upon Leu∙Gly∙Gly have been separated from bovine brain extracts, and three have been highly purified by ammonium sulfate fractionation and by chromatography on DEAE-cellulose, DEAE-Sephadex, and hydroxylapatite. A Mn2+-stimulated aminopeptidase (E2) was purified 180-fold over an initial 16 300 × g supernatant fraction and has a M.W. of approximately 117 250 as estimated by sucrose density gradient centrifugation. Two peptidases which are inhibited by heavy metals (E1P1 and E1P2) were purified approximately 2683-fold and 2461-fold over the initial extracts and have approximate M.W.'s of 61 500 and 85 700, respectively. E1P1 and E1P2 have Km's of 0.36 and 0.38 mM, respectively. The pH optima for E1P1, E1P2, and E2 are 6.6–7.8, 7.2–8.1, and 7.8–9.6. E2 is more heat-labile than E1P1 and E1P2 since it is completely inactivated by heating at 55° for 30 min at pH 6.5 under which conditions the latter two enzymes retain full activity. The ΔEa for the hydrolysis of Leu∙Gly∙Gly was calculated to be 10 800, 13 420, and 15 480 for E1P1, E1P2, and E2. E1P1 and E1P2 hydrolyze tripeptides but do not cleave dipeptides. E2 cleaves tripeptides, and only cleaves Leu∙Gly, of a series of dipeptides examined, having a specificity differing notably from leucine aminopeptidase. All the enzymes were inactive towards leucine-p-nitroanilide and leucine-β-naphthylamide, suggesting that they should not be classed among the arylamidases. On the basis of differential heat stability, it is estimated that a peptidase sedimented by ammonium sulfate at 20% saturation contains 40% of the original tripeptidase activity; E2 represents 30% of the activity; E1P1 and E1P2 contribute 12 and 18%, respectively, to the tripeptidase activity in the original extracts.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

Synapsin-1 is found in a microtubule-associated complex of proteins isolated from bovine brain

1990 ◽  
Vol 68 (11) ◽  
pp. 1256-1261 ◽  
Author(s):  
Karen P. Farrell ◽  
Robert A. B. Keates
Keyword(s):  
Peptide Mapping ◽  
Gradient Centrifugation ◽  
Staining Intensity ◽  
Sucrose Density Gradient ◽  
Bovine Brain ◽  
Sucrose Density Gradient Centrifugation ◽  
Usual Procedure ◽  
Brain Spectrin ◽  
Synapsin 1

We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.Key words: microtubule-associated protein, synapsin, spectrin, tubulin, cytoskeleton.

scholarly journals Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

1975 ◽  
Vol 65 (3) ◽  
pp. 577-586 ◽  
Author(s):  
J Noseworthy ◽  
G H Smith ◽  
S R Himmelhoch ◽  
W H Evans
Keyword(s):  
Guinea Pig ◽  
Polymorphonuclear Leukocytes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Species Difference ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Supernatant Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Electrophoretic Patterns

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.

scholarly journals A novel microtubule-associated protein from mammalian nerve shows ATP-sensitive binding to microtubules.

1986 ◽  
Vol 103 (4) ◽  
pp. 1539-1545 ◽  
Author(s):  
P J Hollenbeck ◽  
K Chapman
Keyword(s):  
Atpase Activity ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Spinal Nerve ◽  
Bovine Brain ◽  
Sucrose Density Gradient Centrifugation ◽  
Associated Proteins ◽  
Free Microtubules ◽  
Weight Protein ◽  
Mammalian Nerve

We report the isolation of a protein from mammalian nerve which shows ATP-sensitive binding to microtubules and ATPase activity. This protein, which we have designated HMW4, was prepared from bovine spinal nerve roots by microtubule affinity and ATP-induced release, and was further purified by sucrose density gradient centrifugation. It is a high molecular weight protein with a denatured Mr of 315,000, a Stokes radius of 90 A, and a sedimentation value of approximately 19S. It can be resolved electrophoretically from the well-characterized bovine brain microtubule-associated proteins (MAPs) and also appears to be distinct from MAP 1C. HMW4 has a vanadate-sensitive and azide-insensitive ATPase activity which averages 20 nmol Pi/min per mg protein and is different from dynein and myosin ATPases. HMW4 prepared on sucrose gradients exhibits binding to MAP-free microtubules in the absence of ATP which is reduced by ATP addition. Assayed by darkfield microscopy, HMW4 causes bundling of MAP-free microtubules which is reversed by ATP addition.

scholarly journals Chemical cross-linking of chick oviduct progesterone-receptor subunits by using a reversible bifunctional cross-linking agent

1979 ◽  
Vol 181 (1) ◽  
pp. 201-213 ◽  
Author(s):  
M E Birnbaumer ◽  
W T Schrader ◽  
B W O'Malley
Keyword(s):  
Progesterone Receptor ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Receptor Proteins ◽  
The Cross

Chick oviduct progesterone-receptor proteins were treated in cytosol with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate. The product of the reaction was a 7S complex that could be detected and recovered after sucrose-density-gradient centrifugation in 0.3M-KCl. The extent of the reaction was dependent on the concentration of methyl 4-mercaptobutyrimidate and independent of the presence of bound hormone, since unlabelled receptors could also be cross-linked. The cross-linking reaction required conditions in which the cytosol 6S complex was preserved. A Stokes radius of 7.3 nm was determined by gel filtration in Agarose A-1.5 m in 0.3 M-KCl. The sedimentation coefficient, which was also determined in 0.3 M-KCl, allowed us to calculate a mol. wt. of 228,000. We were also able to cross-link partially purified receptor forms isolated by using an Agarose A-15 m column. On reduction with beta-mercaptoethanol the complex broke down to 4S monomers that were identified by DEAE-cellulose and phosphocellulose chromatography, adsorption on DNA-cellulose and gel filtration in an Agarose A-1.5 m column. In most cases, A and B receptor proteins were released in equivalent amounts, implying that the cross-linked form was an A-B complex.

Studies on 2′,3′-cyclic nucleotide-3′-phosphohydrolase from brain

1969 ◽  
Vol 47 (10) ◽  
pp. 961-966 ◽  
Author(s):  
Robert W. Olafson ◽  
George I. Drummond ◽  
Jack F. Lee
Keyword(s):  
Density Gradient ◽  
Cyclic Nucleotide ◽  
Gradient Centrifugation ◽  
Physiological Role ◽  
Sucrose Density Gradient ◽  
Bovine Brain ◽  
Sucrose Density Gradient Centrifugation ◽  
Brain White Matter ◽  
Cyclic Phosphate

Studies on a 2′,3′-cyclic nucleotide-3′-phosphohydrolase are presented. Bovine brain white matter was fractionated by differential centrifugation and sucrose density gradient techniques. The phosphohydrolase was distributed throughout all primary fractions: nuclear, mitochondrial, and microsomal. When these fractions were subjected to sucrose density gradient centrifugation the enzyme appeared only in the myelin-containing layers. Brains of newborn rats were essentially devoid of phosphohydrolase activity and the content rose rapidly during the 12th and 25th days, coinciding precisely with the development of myelin. Mutant 'quaking' mice whose brains are partially deficient in myelin were also partially deficient in enzyme activity. The data provide evidence that the phosphohydrolase is associated with myelin.The enzyme opens the cyclic phosphodiester linkage of Ap(Ap)2A cyclic P and Ap(Ap)6A cyclic P without rupture of internucleotide bonds. The enzyme also hydrolyzed the 2′,3′-cyclic phosphorothioates of uridine and guanosine. The Km for adenosine 2′,3′-cyclic phosphate was determined to be 1.9 × 10−3 M, an inordinately high value, and since nothing is known about the physiological role of the enzyme, the possibility is raised that the true substrate may not be a cyclic nucleotide.

scholarly journals Synthesis of deoxyribonucleic acid in BHK-21/C13 cells

1973 ◽  
Vol 131 (3) ◽  
pp. 499-508 ◽  
Author(s):  
Geoffrey J. Hayton ◽  
Colin K. Pearson ◽  
Jeremy R. Scaife ◽  
Hamish M. Keir
Keyword(s):  
Deoxyribonucleic Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Kidney Cells ◽  
Baby Hamster Kidney ◽  
Sucrose Density Gradient Centrifugation ◽  
Mechanism Model ◽  
Baby Hamster Kidney Cells

Newly synthesized 3H-labelled DNA was extracted from baby hamster kidney cells (BHK-21/C13 cells) and was shown to possess single-stranded properties when examined by column chromatography on benzoylated naphthoylated DEAE-cellulose, hydroxyapatite and methylated albumin on kieselguhr, and by its affinity for nitrocellulose filters. Some of the newly synthesized DNA was shown to be of lower molecular weight than the bulk of the DNA when examined by alkaline sucrose-density-gradient centrifugation. The properties observed were not affected by treatment of the DNA with ribonuclease, Pronase or amylase. The effect of the size of the DNA on its observed properties was examined and is discussed. It is concluded that DNA synthesis in BHK-21/C13 cells proceeds according to the discontinuous-mechanism model in at least one of the strands.

Deoxyribonucleic acid polymerase from nuclei of rat intestinal mucosa

1970 ◽  
Vol 48 (5) ◽  
pp. 529-536 ◽  
Author(s):  
F. Y. T. Leung ◽  
S. H. Zbarsky
Keyword(s):  
Dna Polymerase ◽  
Intestinal Mucosa ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Molecular Size ◽  
Sucrose Density Gradient ◽  
Polymerase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Dna Template

An extract with DNA polymerase activity was prepared from nuclei of intestinal mucosa of the rat. Chromatography of the crude extract on DEAE-cellulose yielded three enzymically active fractions: I, II, and III. Each fraction could be resolved further into two components with DNA polymerase activity by rechromatography on smaller columns of DEAE-cellulose. A similar result was obtained by gel filtration of fraction II material on Sephadex G-150. The result of sucrose density gradient centrifugation of the fractions obtained by gel filtration suggested that each still consisted of a mixture of proteins with DNA polymerase activity. The approximate molecular weights of the active proteins, estimated by comparison with marker proteins, ranged from 25 000 to 300 000. Partially purified DNA polymerase (fraction II) required for activity the four deoxyribonucleoside triphosphates, Mg2+, 2-mercaptoethanol, and DNA template. The optimum pH for activity was 8.0 in Tris–HCl buffer and 7.4 in phosphate buffer. The two components obtained by gel filtration of fraction II differed in their requirements for DNA template. The one of smaller molecular size was more active with native DNA whereas the larger was equally active with either native or heat-denatured DNA.

scholarly journals Uptake of oestradiol by the rabbit hypothalamus. Specificity of binding by nuclei in vitro

1970 ◽  
Vol 118 (1) ◽  
pp. 93-97 ◽  
Author(s):  
Gerald J. Chader ◽  
Claude A. Villee
Keyword(s):  
Specific Binding ◽  
Gradient Centrifugation ◽  
Significant Proportion ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Cerebellar Nuclei ◽  
Supernatant Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Hypothalamic Nuclei

The binding of [6,7-3H]oestradiol-17β to subcellular fractions of the hypothalamus and the cerebellum of the rabbit was studied in vitro. Uptake of steroid was higher in hypothalamic nuclei than in cerebellar nuclei. Lower binding was observed in other fractions of both tissues. After dialysis of the fractions, hypothalamic nuclei retained a high percentage of oestradiol whereas cerebellar nuclei lost most of the bound steroid. Supernatant fractions of both tissues retained a significant proportion of label after dialysis and after gel filtration on Sephadex G-200. No specific binding was observed in these fractions when subjected to sucrose-density-gradient centrifugation. Purification of nuclei followed by incubation with labelled oestradiol in the absence of the supernatant fraction resulted in loss of binding of steroid by hypothalamic nuclei. Incubation of the purified hypothalamic nuclei with supernatant fraction maintained the binding specificity of hormone retention.

Apparent saturability of a 4S dihydrotestosterone-binding protein in rat muscle cytosol: role of 3α-hydroxysteroid dehydrogenase and albumin

1977 ◽  
Vol 55 (9) ◽  
pp. 995-1000 ◽  
Author(s):  
France T. Dionne ◽  
Jean Y. Dube ◽  
Roland R. Tremblay
Keyword(s):  
Enzymatic Activity ◽  
Ammonium Sulfate ◽  
Binding Protein ◽  
Gradient Centrifugation ◽  
Hydroxysteroid Dehydrogenase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Rat Serum ◽  
Binding Behaviour

In our studies of the binding of steroids in rat skeletal muscles, we have shown the existence of a saturable '4S' dihydrotestosterone-binding protein of low affinity (Kd = 1.16 × 10−6 M). Competition studies led us to believe that the binding of dihydrotesterone (DHT) was due to 3α-hydroxysteroid dehydrogenase (3α-OHSD) enzyme responsible for the conversion of DHT to androstanediol (Adiol). Indeed, both the binding and the enzymatic activity are inhibited by various 3-keto steroids. In addition, the dissociation constant (Kd) and the Michaelis constant (Km) of the enzyme are similar. However, experiments with ammonium sulfate fractions of the cytosol have shown a partial separation of the binding and of the enzymatic activity. On the other hand, we have established that DHT (3 μM) is almost completely metabolized to Adiol during a 2-h incubation at 0 °C even in the absence of added coenzymes. Furthermore the '4S' protein binds Adiol more strongly than DHT and this binding is not saturable. Finally the binding behaviour of both DHT and Adiol with either muscle cytosol or rat albumin is similar when subjected to ammonium sulfate fractionation and sucrose density gradient centrifugation. In conclusion, the skeletal muscle 3α-OHSD rapidly metabolizes DHT into Adiol which then binds strongly to a nonspecific binding protein, presumably rat serum albumin. Thus it can be said that the observed saturability of DHT binding is only apparent.

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