Role of vacuoles and vesicles in extracellular enzyme secretion from yeast

1973 ◽  
Vol 19 (1) ◽  
pp. 113-117 ◽  
Author(s):  
R. A. Holley ◽  
D. K. Kidby
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Extracellular Enzyme ◽  
Invertase Activity ◽  
Preliminary Evidence ◽  
Acid Protease ◽  
Intracellular Enzyme ◽  
Per Se ◽  
Equilibrium Centrifugation

Preliminary evidence has been obtained which suggests that the intracellular invertase of Saccharomyces cerevisiae may not be localized in the vacuole per se. Alkaline phosphatase, an intracellular enzyme, and acid protease, a typically lysosomal enzyme, both showed high specific activity in the vacuole fraction prepared by equilibrium centrifugation of lysed sphaeroplasts in Ficoll gradients. Invertase activity has been found to be associated with vacuoles only when glucose-repressed cells are derepressed. Cells derepressed for invertase biosynthesis contained a population of vesicles which were virtually absent from the repressed cells. Evidence is presented which strongly suggests that these vesicles rather than the vacuoles are the vehicle by which invertase is secreted from the cell.

Genotype/Phenotype Correlations for Coagulation Factor XIII: Specific Normal Polymorphisms Are Associated With High or Low Factor XIII Specific Activity

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 897-905 ◽  
Author(s):  
Rashida Anwar ◽  
Louise Gallivan ◽  
Stuart D. Edmonds ◽  
Alexander F. Markham
Keyword(s):  
Factor Xiii ◽  
Specific Activity ◽  
Recurrent Miscarriage ◽  
Coagulation Factor ◽  
Preliminary Evidence ◽  
Activity Levels ◽  
Normal Hemostasis ◽  
Fxiii Activity ◽  
Normally Distributed

Abstract Factor XIII is a transglutaminase essential for normal hemostasis. We have studied the plasma FXIII levels and FXIII activity in 71 individuals and found these to be normally distributed. FXIII specific activity is also normally distributed. However, we show that FXIII activity is not directly dependent on FXIII levels, and individuals with low FXIII levels may have high FXIII activity and vice versa. We have determined the FXIIIA genotype in these individuals to assess whether the variation observed in FXIII specific activity is dependent on specific polymorphisms in the FXIIIA gene. Our data show that the Leu34 and Leu564 variants give rise to increased FXIII specific activity, while the Phe204 variant results in lower FXIII specific activity. We also report preliminary evidence that the Phe204 polymorphism may be associated with recurrent miscarriage. Overall, we have identified 23 unique FXIIIA genotypes. Certain specific FXIIIA genotypes consistently give rise to high, low, or median FXIII specific activity levels, while others appear to have little or no consistent influence on the FXIII phenotype. These genotype to phenotype relationships are discussed in light of the growing interest in the role of FXIII in clinical problems involving an increased thrombotic tendency.

scholarly journals Both Subunits of ATP-Citrate Lyase from Chlorobium tepidum Contribute to Catalytic Activity

2006 ◽  
Vol 188 (18) ◽  
pp. 6544-6552 ◽  
Author(s):  
Wonduck Kim ◽  
F. Robert Tabita
Keyword(s):  
Catalytic Mechanism ◽  
Specific Activity ◽  
High Specific Activity ◽  
Small Subunit ◽  
High Yield ◽  
Chlorobium Tepidum ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Green Sulfur

ABSTRACT ATP-citrate lyase (ACL) is an essential enzyme of the reductive tricarboxylic acid (RTCA) pathway of CO2 assimilation. The RTCA pathway occurs in several groups of autotrophic prokaryotes, including the green sulfur bacteria. ACL catalyzes the coenzyme A (CoA)-dependent and MgATP-dependent cleavage of citrate into oxaloacetate and acetyl-CoA, representing a key step in the RTCA pathway. To characterize this enzyme from the green sulfur bacterium Chlorobium tepidum and determine the role of its two distinct polypeptide chains, recombinant holo-ACL as well as its two individual subunit polypeptides were synthesized in Escherichia coli. The recombinant holoenzyme, prepared from coexpressed large and small ACL genes, and the individual large and small subunit polypeptides, prepared from singly expressed genes, were all purified to homogeneity to high yield. Purified recombinant holo-ACL was isolated at high specific activity, and its k cat was comparable to that of previously prepared native C. tepidum ACL. Moreover, the purified recombinant large and small subunit polypeptides were able to reconstitute the holo-ACL in vitro, with activity levels approaching that of recombinant holo-ACL prepared from coexpressed genes. Stoichiometric amounts of each subunit protein were required to maximize the activity and form the most stable structure of reconstituted holo-ACL. These results suggested that this reconstitution system could be used to discern the catalytic role of specific amino acid residues on each subunit. Reconstitution and mutagenesis studies together indicated that residues of each subunit contributed to different aspects of the catalytic mechanism, suggesting that both subunit proteins contribute to the active site of C. tepidum ACL.

scholarly journals Localization of sites of enhanced expression of endothelin-1 in the kidney of DOCA-salt hypertensive rats.

1996 ◽  
Vol 7 (8) ◽  
pp. 1158-1164 ◽  
Author(s):  
L Y Deng ◽  
R Day ◽  
E L Schiffrin
Keyword(s):  
Mesangial Cells ◽  
Specific Activity ◽  
High Specific Activity ◽  
Hypertensive Rats ◽  
Endothelin 1 ◽  
Renal Vessels ◽  
Enhanced Expression ◽  
Potent Vasoconstrictor

Although the role of endothelin-1, a potent vasoconstrictor peptide, in hypertension remains unclear, there is evidence of its involvement in deoxycorticosterone acetate (DOCA)-salt hypertensive rats, in which enhanced vascular production of endothelin-1 has been documented. The study presented here examined endothelin-1 gene expression in the kidney in DOCA-salt hypertensive rats by in situ hybridization histochemistry. A high specific activity 35S-labeled complementary RNA probe was used. Significant increases in abundance of endothelin-1 mRNA transcripts were found in the endothelium of renal vessels, and in capillary endothelial and mesangial cells of glomeruli of the remaining kidney of DOCA-salt hypertensive rats, in comparison with unilaterally nephrectomized control rats. Enhanced expression of the endothelin-1 gene in the kidney of DOCA-salt hypertensive rats may participate in abnormalities of renal function in this model of hypertension, and thus contribute to the development and maintenance of elevated blood pressure.

scholarly journals Possible role of growth regulators in adaptation to heat stress affecting partitioning of photosynthates in tomato plants

2014 ◽  
Vol 58 (1) ◽  
pp. 71-84 ◽  
Author(s):  
Zofia Starck ◽  
Elżbieta Cieśla
Keyword(s):  
Heat Stress ◽  
High Temperature ◽  
Growth Regulators ◽  
Specific Activity ◽  
Invertase Activity ◽  
Acid Invertase ◽  
Sink Strength ◽  
Tomato Plants ◽  
Different Response

Tomato plants of two cultivars: Roma - sensitive and Robin - tolerant to heat stress were grown in greenhouse up to the flowering stage and then under controlled environmen­tal conditions. The partitioning of recently fixed <sup>14</sup>CO<sub>2</sub> by mature tomato leaves was examined as a posteffect of 24-h heat stress (38/25°C day/night) with the interaction of growth regulators (GR) sprayed on the flowers with solution of β-naphthoxyacetic (NOA) and gibberellic (GA<sub>3</sub>) acid (denoted as NG), or Zeatin + NOA + GA<sub>3</sub> (denoted as ZNG). In both cuitivars GR strongly stimulated fruit growth and transport of <sup>14</sup>C-photosynthates to the clusters at the expense of vegetative organs. Heat stress decreased export of <sup>14</sup>C-phoiosynthates from the blades in plants not treated with GR, but even more in cv. Roma. In Roma plants not treated with GR (with very small fruitlets and fruits) the heat stress retarded <sup>14</sup>C-transport just in the petioles, diminishing the <sup>14</sup>C-supply to the fruits. Reduction of the current photosynthate supplied to the fruits seems to be causally connected with inhibition of the specific activity of acid invertase in that organ. Growth regulators reduced the negative effect of high temperature - they alleviated depression of <sup>14</sup>C-export from the blades and increased invertase activity. <sup>14</sup>C-photosynthate transport to the fruits, presumably owing to their higher sink strength, was less affected by heat stress. In Robin plants (which had bigger fruits during the experiment) high temperature depressed <sup>14</sup>C-fruit supply only in the NG-series, in contrast to enhacement of <sup>14</sup>C-Movement to that sink in the control and ZNG-series. In spite of these facts, after heat stress, the specific activity of acid invertase decreased in all the experimental series, but much less in the GR-treated series. Therefore, in the Robin cv. there was no relation between invertase activity and <sup>14</sup>C-mobilization by fruits, as was observed in Roma plants. The possible explanation of the different response of the two cultivars with contrasting sensitivity to heat stress; with special reference to the role of GR; diminishing injury of the plants by high temperature is discussed.

Role of pancreatic L-asparagine synthetase in homeostasis of L-asparagine.

1979 ◽  
Vol 236 (6) ◽  
pp. E746 ◽  
Author(s):  
H A Milman ◽  
D A Cooney ◽  
D M Young
Keyword(s):  
Amino Acid ◽  
Exocrine Pancreas ◽  
Specific Activity ◽  
Pancreatic Enzyme ◽  
High Specific Activity ◽  
Asparagine Synthetase ◽  
Mouse Pancreas ◽  
Normal Limits ◽  
Intravenous Treatment

L-Asparagine synthetase from mouse pancreas was found to be associated principally with the exocrine pancreas and to be dependent on the age of the animal, but not on gender, diet, or the presence of tumor under the conditions examined. The function of the pancreatic enzyme appears to be to supply L-asparagine for the synthesis of pancreatic proteins. This function is suggested by the high specific activity of L-asparagine in pancreatic proteins after intravenous treatment of BDF1 mice with L-[U-14C]asparatate. The pancreas is also able to function as a storage depot for L-asparagine under conditions in which the concentration of the amino acid in the blood is in excess. Unlike the liver, the pancreas is unable to add L-asparagine to the circulation when the concentration of the amide is below normal limits.

Are Spinach Chloroplasts Involved in Flavonoid O-Methylation?

1985 ◽  
Vol 40 (5-6) ◽  
pp. 331-335 ◽  
Author(s):  
Kim Thresh ◽  
Ragai K. Ibrahim
Keyword(s):  
Specific Activity ◽  
Specific Binding ◽  
Plant Secondary Metabolites ◽  
High Specific Activity ◽  
Spinach Chloroplasts ◽  
Comt Activity ◽  
Activity Ratios ◽  
Methylation Patterns ◽  
Spinach Leaves

Abstract Intact spinach chloroplasts, which were isolated on a cushion of Percoll and purified on discon­tinuous sucrose gradients, were found to adsorb appreciable amounts of high specific activity, cytosolic O-methyltransferases. The latter activity was readily eliminated after washing the chloroplast pellet with 0.1 ᴍ phosphate in sucrose gradient buffer and finally with 0 .1% nonidet. Furthermore, the OMT activity of spinach leaves was resolved by chromatography on DEAE-Sepharose CL-6B into caffeic acid (COMT) and flavonoid (FOMT) O-methyltransferase ac­tivities, and purified to 30-and 50-fold, respectively. The similarities between the FOMT/COMT activity ratios and the methylation patterns of intact chloroplasts and their supernatants, as well as those of purified leaf preparations, suggest non-specific binding of cytosolic OMTs to chloroplast envelopes. It is concluded, therefore, that spinach chloroplasts are not involved in the methylation of phenylpropanoid or flavonoid compounds. These results call attention for the re-evaluation of the role of this organelle in the biosynthesis or accumulation of plant secondary metabolites.

Role of anions and carbonic anhydrase in epithelia

1982 ◽  
Vol 299 (1097) ◽  
pp. 369-381 ◽  
Keyword(s):  
Carbonic Anhydrase ◽  
Epithelial Transport ◽  
Carbonic Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
Salt Transport ◽  
Biliary Duct ◽  
Toad Urinary Bladder ◽  
Hydration And Dehydration

The existence of carbonic anhydrase (carbonate dehydratase, EC 4. 2.1.1) in blood was suspected and sought because the rates of spontaneous hydration and dehydration of CO 2 and carbonic acid were slow compared with the rates of exchange of CO 2 with blood. The existence of the enzyme in absorbing and secreting epithelial tissues has, in contrast, often been sought because its presence was required for the operations of theoretical models for the movements of H + ions or HCO - 3 into or out of epithelial cells. In addition to the HCl-secreting gastric mucosal epithelium, the enzyme was subsequently found in the rumen, in the kidney, especially those of species that produce acid urine, in salivary gland, the liver and biliary duct system, the mucosa of the small intestine, caecum and colon, the choroid plexuses and ciliary body of mammals, in toad urinary bladder and in the Cl - secreting cells of fish gill. The presence of carbonic anhydrase in exocrine pancreas does not seem to be well established. The enzyme, of molecular mass about 30 kDa and containing one zinc atom, exists in three related forms: one of high specific activity and two of low specific activity, one of which is found in red skeletal muscle. Although most, but not all, types of erythrocyte contain both varieties, epithelia usually contain only the highactivity enzyme; however, ox rumen contains large quantities of the low-activity variety as do guinea-pig caecal and colonic mucosae. Salt transport in the intestinal tract is associated with movements of HCO - 3 and of H + ions, yet although carbon dioxide stimulates solute and fluid transport in the gall bladder and jejunum, and inhibitors of carbonic anhydrase reduce fluid and ion transport across many epithelia, the role of the enzyme in epithelial transport is not clearly understood. Knowledge of the rates of hydration and dehydration of CO 2 /HCO - 3 in the fraction of the tissue water responsible for the H + -HCO - 3 movements in many secretory epithelia is currently lacking.

Genotype/Phenotype Correlations for Coagulation Factor XIII: Specific Normal Polymorphisms Are Associated With High or Low Factor XIII Specific Activity

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 897-905 ◽  
Author(s):  
Rashida Anwar ◽  
Louise Gallivan ◽  
Stuart D. Edmonds ◽  
Alexander F. Markham
Keyword(s):  
Factor Xiii ◽  
Specific Activity ◽  
Recurrent Miscarriage ◽  
Coagulation Factor ◽  
Preliminary Evidence ◽  
Activity Levels ◽  
Normal Hemostasis ◽  
Fxiii Activity ◽  
Normally Distributed

Factor XIII is a transglutaminase essential for normal hemostasis. We have studied the plasma FXIII levels and FXIII activity in 71 individuals and found these to be normally distributed. FXIII specific activity is also normally distributed. However, we show that FXIII activity is not directly dependent on FXIII levels, and individuals with low FXIII levels may have high FXIII activity and vice versa. We have determined the FXIIIA genotype in these individuals to assess whether the variation observed in FXIII specific activity is dependent on specific polymorphisms in the FXIIIA gene. Our data show that the Leu34 and Leu564 variants give rise to increased FXIII specific activity, while the Phe204 variant results in lower FXIII specific activity. We also report preliminary evidence that the Phe204 polymorphism may be associated with recurrent miscarriage. Overall, we have identified 23 unique FXIIIA genotypes. Certain specific FXIIIA genotypes consistently give rise to high, low, or median FXIII specific activity levels, while others appear to have little or no consistent influence on the FXIII phenotype. These genotype to phenotype relationships are discussed in light of the growing interest in the role of FXIII in clinical problems involving an increased thrombotic tendency.

Validation of a sensitive direct assay for melatonin for investigation of circadian rhythms in different species

1985 ◽  
Vol 106 (3) ◽  
pp. 387-394 ◽  
Author(s):  
G. E. Webley ◽  
H. Mehl ◽  
K. P. Willey
Keyword(s):  
Specific Activity ◽  
Biological Fluids ◽  
High Specific Activity ◽  
Male Rat ◽  
Rat Serum ◽  
Menstrual Cycles ◽  
High Affinity ◽  
Direct Assay ◽  
Wide Range

ABSTRACT The role of melatonin in animals which do not show marked seasonal changes in reproduction is disputed, in part because of the wide variation in reported concentrations. One reason for this may be the difficulties associated with the measurement of low molar concentrations of melatonin and the presence of a wide variety of potentially cross-reacting substances. The availability of a high affinity antiserum has allowed an assay, with low cross-reactivity and good sensitivity, to be established for the direct measurement of melatonin in a wide range of biological fluids, in particular serum, plasma and follicular fluid from man and rat. The high affinity of the antiserum enabled a tritium label of high specific activity to be used, removing the problems associated with the iodination of a small molecular weight compound. Melatonin concentrations in the assay were evaluated by four different methods: UV absorbance, gas chromatography, comparison of the immunoreactive concentrations of the label with the expected concentration by dilution and by comparison with a previously established assay which uses the same antiserum. Melatonin was measured in serum from twelve healthy women over two 24-h periods; eight women with normal menstrual cycles and four taking the contraceptive pill. Concentrations were found to range from 19·8 to 215 pmol/l during the day in both groups. In women with normal menstrual cycles peak concentrations of 513·2 ± 54·1 (s.e.m.) pmol/l were recorded at 04.00 h, whereas higher concentrations were found in women taking the pill, reaching a peak of 849·12 ± 21·8 (s.e.m.) pmol/l at 04.00 h. Similar melatonin concentrations were measured in the two 24-h periods. In the adult male rat, serum melatonin concentrations varied from 92·66 ± 37·9 (s.e.m.) pmol/l at 12.00 h, rising to 526 ± 55·6 (s.e.m.) pmol/l at 04.00 h. This direct assay is more practical and robust than the assays currently available. The careful validation of assay characteristics allows its widespread use in both clinical studies and the investigation of the role of melatonin in different species. J. Endocr. (1985) 106, 387–394

scholarly journals Heterogeneous phosphorylation of erythrocyte spectrin β chain in intact cells

1993 ◽  
Vol 294 (3) ◽  
pp. 841-846 ◽  
Author(s):  
S Pedroni ◽  
M C Lecomte ◽  
H Gautero ◽  
D Dhermy
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Beta Chain ◽  
Intact Cells ◽  
Molecular Masses ◽  
Alpha Beta ◽  
Self Association

Human erythrocyte spectrin is an alpha beta heterodimer which forms tetramers by self-association. This association involves the N-terminal region of the alpha chain and the C-terminal region of the beta chain. The latter contains a cluster of four phosphorylation sites (one phosphothreonine and three phosphoserine residues). The role of this phosphorylation is as yet unknown. We show in this paper that the spectrin beta chain occurs in the cell in subpopulations differing in the degree of occupancy of their phosphorylation sites: 32P peptide maps obtained by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage revealed the presence of six components with apparent molecular masses of 17.5 kDa, differing in their isoelectric points; this is most simply interpreted as reflecting the presence of six exchangeable phosphorylation sites in the spectrin beta chain, rather than four as had been supposed. When the alpha beta dimers were partly dissociated by urea, the most highly phosphorylated fraction of the beta chain was found in the undissociated dimers. This high specific activity in the undissociated dimer reflected multiple phosphorylated sites, as revealed by NTCB cleavage. The dephosphorylation or the hyperphosphorylation of spectrin beta chains did not modify the equilibrium between dissociated and undissociated spectrin dimers in the presence of urea. However, the data revealed the existence of two spectrin dimer populations in respect to phosphate turnover and spectrin dimer dissociation.

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