scholarly journals Protease and ribonuclease activities in bovine pituitary lobes

1971 ◽  
Vol 123 (2) ◽  
pp. 153-162 ◽  
Author(s):  
J. C. Pickup ◽  
D. B. Hope
Keyword(s):  
Trichloroacetic Acid ◽  
Posterior Pituitary ◽  
Neurosecretory Granules ◽  
Alkaline Ph ◽  
Inhibitory Effects ◽  
Ph Optimum ◽  
Exogenous Rna ◽  
Absorbing Material ◽  
Endogenous Substrate ◽  
Alkaline Ribonuclease

1. Acid and alkaline protease activities in bovine anterior and posterior pituitary lobes were reinvestigated by measurement of u.v. and Folin–Ciocalteu colour values of trichloroacetic acid-soluble digestion products of denatured haemoglobin. 2. Both lobes of the pituitary gland contain a cathepsin with a pH optimum at 3.8. 3. When release of u.v.-absorbing material was used as the assay there was also an optimum at pH8.3–9.7, but this proved to be due to the release of nucleosides from an endogenous substrate. 4. The presence of a `cyclizing' ribonuclease active at alkaline pH on endogenous RNA was confirmed by the inhibitory effects of phosphate, arsenate and bentonite. The activity was unaffected by heat, EDTA or metal ions. The enzyme also acted on exogenous RNA. 5. A purified preparation of neurosecretory granules from fresh bovine posterior pituitary lobes was free from alkaline ribonuclease activity. Most of the activity present in the tissue was recovered in the supernatant plus microsomal material. 6. The distribution of RNA did not follow that of the alkaline ribonuclease.

scholarly journals Degredation of insulin by a particulate fraction from adipose tissue

1970 ◽  
Vol 116 (5) ◽  
pp. 825-831 ◽  
Author(s):  
Kenneth Sumner ◽  
Richard J. Doisy
Keyword(s):  
Adipose Tissue ◽  
Paper Chromatography ◽  
Trichloroacetic Acid ◽  
Degradation Products ◽  
Enzyme System ◽  
Enzyme Concentration ◽  
Particulate Fraction ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Rat Adipose Tissue

The destruction of 125I-labelled insulin by an enzyme system from rat adipose tissue was studied. The system was located in the particulate fraction. Activity was assayed by the amount of 125I-labelled degradation products rendered soluble in trichloroacetic acid. The system was heat-labile, with an alkaline pH optimum. The velocity of the reaction varied directly with the enzyme concentration. Paper chromatography of the degradation products showed six ninhydrin-sensitive areas with radioactivity coinciding with three of them. Albumin inhibited the system; ribonuclease did not. Although only 25% of the total 125I-label was detected by this assay, results with insulin-specific assays suggested that most (80–90%) of the hormone was inactivated. Possible interpretations of these results are discussed. The particulate fractions of other tissues were also studied.

PHOSPHODIESTERASE AND ACID PHOSPHATASE ACTIVITIES OF AVIAN BONE MARROW CELLS

1965 ◽  
Vol 43 (8) ◽  
pp. 1319-1328 ◽  
Author(s):  
Gerald Kingsley Bristow ◽  
Esther W. Yamada
Keyword(s):  
Bone Marrow ◽  
Acid Phosphatase ◽  
Bone Marrow Cells ◽  
Intracellular Distribution ◽  
Specific Substrate ◽  
Magnesium Ions ◽  
Differential Centrifugation ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Rna And Dna

Avian bone marrow has been found to contain a phosphodiesterase as well as an acid phosphatase. Some properties of these enzymes have been described. Because the phosphodiesterase of this tissue has an alkaline pH optimum, is activated by magnesium ions, and acts on the specific substrate p-nitrophenyl thymidine 5′-phosphate, it is probably a phosphodiesterase I such as is present in snake venom and other tissues.The intracellular distribution of these two enzymes in normal and regenerating bone marrow was studied. Subcellular fractions were prepared by differential centrifugation or by centrifugation through sucrose density gradients. The RNA and DNA content of each fraction was determined. By the methods used no differences in the properties or intracellular distribution of the two enzymes in normal and regenerating bone marrow were found.

scholarly journals Subcellular organization of neurophysins, oxytocin, [8−lysine]-vasopressin and adenosine triphosphatase in porcine posterior pituitary lobes

1973 ◽  
Vol 132 (3) ◽  
pp. 361-371 ◽  
Author(s):  
J. C. Pickup ◽  
C. I. Johnston ◽  
S. Nakamura ◽  
L. O. Uttenthal ◽  
D. B. Hope
Keyword(s):  
Density Gradient ◽  
Adenosine Triphosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Posterior Pituitary ◽  
Neurosecretory Granules ◽  
Sucrose Density Gradient Centrifugation ◽  
Lysine Vasopressin ◽  
Neurophysin Ii

Posterior pituitary lobes from young pigs were fractionated by differential and sucrose-density-gradient centrifugation. The distributions of oxytocin and [8-lysine]-vasopressin were measured by bioassay and the distributions of neurophysin-I and -II by radioimmunoassays specific for each of these two proteins. Most of the hormone and neurophysin applied to the density gradient was localized in particles with the density expected of neurosecretory granules. However, the neurosecretory granules were separated into two bands (D and E). A close statistical correlation between the distributions of [8-lysine]-vasopressin and neurophysin-I, and of oxytocin and neurophysin-II on the gradients, suggested that in vivo porcine neurophysin-I binds [8-lysine]-vasopressin within one population of granules and porcine neurophysin-II binds oxytocin within another type of granule. However, there was no significant separation of oxytocin and vasopressin in fractions D and E. The molar ratios of hormones and neurophysins indicated that there was insufficient of either neurophysin to bind the [8-lysine]-vasopressin in the granule fractions or in the whole gland. Polyacrylamide-gel electrophoresis showed that only bands corresponding in mobility to porcine neurophysins-I, -II and -III were present in large amounts in the whole gland and in the granule fractions. The component with the mobility of neurophysin-III was, however, relatively enriched in whole young glands and granule fractions compared with adult gland extracts. It is suggested that the vasopressin that cannot be assigned to neurophysin-I may occur in (a) vesicles containing vasopressin but no neurophysin, (b) vesicles containing vasopressin and a protein that cannot be quantified by the radioimmunoassays used, such as porcine neurophysin-III, or (c) normal vasopressin–neurophysin granules which have accumulated extra vasopressin. Band E of the gradient was rich in adenosine triphosphatase activity, whereas band D possessed very little of this enzyme.

Zebrafish tyrosylprotein sulfotransferase: molecular cloning, expression, and functional characterization

2004 ◽  
Vol 82 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Emi Mishiro ◽  
Ming-Yih Liu ◽  
Yoichi Sakakibara ◽  
Masahito Suiko ◽  
Ming-Cheh Liu
Keyword(s):  
Molecular Cloning ◽  
Divalent Cations ◽  
Functional Characterization ◽  
The Other ◽  
Inhibitory Effects ◽  
Amino Acid Sequence Level ◽  
Ph Optimum ◽  
Rapid Amplification ◽  
High Degree

By employing the reverse transcriptase – polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.Key words: zebra fish, tyrosylprotein sulfotransferase, molecular cloning.

Characterization of a novel insect digestive DNase with a highly alkaline pH optimum

2002 ◽  
Vol 32 (3) ◽  
pp. 255-263 ◽  
Author(s):  
Johann P Schernthaner ◽  
Ross E Milne ◽  
Harvey Kaplan
Keyword(s):  
Alkaline Ph ◽  
Ph Optimum

scholarly journals ISOLATION OF NERVE ENDINGS FROM THE POSTERIOR PITUITARY GLAND

1965 ◽  
Vol 25 (3) ◽  
pp. 179-193 ◽  
Author(s):  
Frank S. Labella ◽  
Madhu Sanwal
Keyword(s):  
Electron Microscopic Examination ◽  
Basement Membranes ◽  
Nerve Endings ◽  
Posterior Pituitary ◽  
Neurosecretory Granules ◽  
Electron Microscopic ◽  
Free Nerve Endings ◽  
Pituitary Glands ◽  
Posterior Pituitary Gland ◽  
Staining Techniques

Bovine posterior pituitary glands were homogenized in 10 per cent sucrose and fractionated by differential centrifugation. The following centrifugation procedure resulted in the most satisfactory separation: 1000 g for 15 minutes—nuclei, connective tissue, basement membranes with associated endothelium, giant nerve endings, and whole pituicytes; 4200 g for 15 minutes—free nerve endings, including Herring bodies; 17,000 g for 15 minutes—mitochondria; 68,000 g for 15 minutes—neurosecretory granules. Electron microscopic examination was carried out on whole tissue and on the isolated fractions. Isolated nerve endings were examined also by negative staining techniques. Isolated nerve endings retain an apparently normal complement of mitochondria, neurosecretory granules, and microvesicles ("synaptic" vesicles). The free nerve endings closely resemble those observed in sections of intact posterior pituitary tissue. Free microvesicles were not observed in any of the fractions isolated and apparently sediment at centrifugal forces higher than those employed in this study.

scholarly journals Effect of microtubular or translational inhibitors on general cell protein degradation. Evidence for a dual catabolic pathway

1977 ◽  
Vol 168 (2) ◽  
pp. 223-227 ◽  
Author(s):  
J S Amenta ◽  
M J Sargus ◽  
F M Baccino
Keyword(s):  
Protein Degradation ◽  
Trichloroacetic Acid ◽  
Fold Increase ◽  
Cell Protein ◽  
Inhibitory Effects ◽  
Minimal Essential Medium ◽  
Catabolic Pathway ◽  
Rat Embryo ◽  
Step Down ◽  
Observation Period

Rat embryo fibroblasts were grown in Eagle's minimal essential medium with 10% serum and cell proteins prelabelled with L-[1-(14)C]leucine, followed by a 24h chase. When transferred to medium deprived of serum these cells showed a 2—3-fold increase in the production of trichloroacetic acid-soluble radioactivity during a 4h observation period. The microtubular poisons vinblastine, vincristine and colchicine partially inhibited this induced proteolysis, but had no effect on the proteolytic rate of cells maintained in medium with 10% serum. A similar discriminating effect on induced proteolysis was observed with cycloheximide, puromycin and insulin. The inhibitory effects of cycloheximide and vinblastine were not additive. These data support the hypothesis that, in addition to the basal turnover of cell proteins, a second mechanism of protein degradation involving cytoplasmic autophagy can be activated by nutritional step-down and is selectively inhibited by agents that interfere with microtubular function and protein synthesis.

scholarly journals Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant

2001 ◽  
Vol 356 (1) ◽  
pp. 19 ◽  
Author(s):  
Dieter BECKER ◽  
Christophe BRAET ◽  
Harry BRUMER III ◽  
Marc CLAEYSSENS ◽  
Christina DIVNE ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Alkaline Ph ◽  
Ph Optimum

Intracellular poly(3-hydroxybutyrate) depolymerase inAlcaligenes eutrophus

1995 ◽  
Vol 41 (13) ◽  
pp. 187-191 ◽  
Author(s):  
Terumi Saito ◽  
Kazuaki Takizawa ◽  
Haruhisa Saegusa
Keyword(s):  
Alcaligenes Eutrophus ◽  
Cell Extract ◽  
Supernatant Fraction ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Ph Profile ◽  
Phb Depolymerase ◽  
Diisopropyl Fluorophosphate ◽  
Triton X ◽  
Two Peaks

The intracellular depolymerization of poly(3-hydroxybutyrate) (PHB) in Alcaligenes eutrophus was investigated. PHB granules of A. eutrophus released D-(−)-3-hydroxybutyrate when incubated at 37 °C. The pH profile of autodigestion of the PHB granules revealed two peaks of activity, one at pH 6–7 and the other at a more alkaline pH. Autodigestion of native PHB granules was inhibited in the presence of Triton X-100. The PHB depolymerase activity was detected in the supernatant from centrifugation at 100 000 × g of the cell extract by using the protease-treated native PHB granules as substrate that lost most of their autodigestive activity. The soluble PHB depolymerase activity increased by about 20-fold or more during PHB synthesis compared with that of PHB-deficient cells, and there was a stringent correlation between PHB content and PHB depolymerase activity in A. eutrophus cells. A soluble PHB depolymerase was partially purified and was inhibited strongly in the presence of diisopropyl fluorophosphate. The pH optimum of the PHB depolymerase in the supernatant fraction was about 8–9. These results indicate that two types of PHB depolymerase may be associated with the native granules, and there was an enzyme with an alkaline pH optimum in the supernatant of the cell extract that seemed to be coregulated with PHB synthesis in the cells.Key words: Alcaligenes eutrophus, poly(3-hydroxybutyrate) depolymerase, poly(3-hydroxybutyrate) granules, biodegradation.

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