scholarly journals Purification of pancreas type-I ATP diphosphohydrolase and identification by affinity labelling with the 5′-p-fluorosulphonylbenzoyladenosine ATP analogue

1995 ◽  
Vol 312 (2) ◽  
pp. 351-356 ◽  
Author(s):  
J Sévigny ◽  
Y P Côté ◽  
A R Beaudoin
Keyword(s):  
Specific Activity ◽  
Biochemical Properties ◽  
Zymogen Granule ◽  
Type I ◽  
Strong Reaction ◽  
Significant Similarity ◽  
Atp Diphosphohydrolase ◽  
Granule Membrane ◽  
Affinity Labelling ◽  
Molecular Masses

The enzyme recently identified as type-I ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) has been purified from the zymogen granule membrane of pig pancreas. After solubilization with Triton X-100 and chromatographies on ion-exchange and Affi-Gel Blue columns an approximate 3500-fold purification was obtained. The enzyme preparation with a specific activity of 45 units/mg of protein was much further purified by PAGE under non-denaturing conditions. The active band localized on the gel contained two proteins after SDS/PAGE and silver staining, corresponding to apparent molecular masses of 56 and 54 kDa. The identity of the ATPDase was confirmed by an affinity labelling technique with 5′-p-fluorosulphonylbenzoyladenosine (FSBA) as an ATP analogue. The latter was detected by a Western blot technique. A strong reaction was observed with the band corresponding to 54 kDa. N-terminal sequence analysis revealed that the 56 kDa protein has significant similarities (50-72%) with lipases, whereas the 54 kDa enzyme has no significant similarity with any known proteins. N-glycosidase F treatment confirmed the glycoprotein nature of the enzyme and suggested that the enzyme bears several N-glycosylation sites. Comparisons of molecular masses and biochemical properties show that this ATPDase is different from other reported mammalian ATPDases.

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[α-32P]ATP

1986 ◽  
Vol 64 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Denis LeBel ◽  
Marlyne Beattie
Keyword(s):  
Binding Sites ◽  
Divalent Cations ◽  
Photoaffinity Labeling ◽  
Competitive Inhibitor ◽  
Atp Binding ◽  
Zymogen Granule ◽  
Atp Diphosphohydrolase ◽  
High Affinity ◽  
Granule Membrane ◽  
Triton X

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[α-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 μM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 μM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A–Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-β-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.

scholarly journals Evidence for an apical, nonregulated protein secretion in pig exocrine pancreas

1994 ◽  
Vol 267 (5) ◽  
pp. G764-G771
Author(s):  
G. Viau ◽  
J. Laine ◽  
F. Levenez ◽  
A. M. Gueugneau ◽  
T. Corring ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Exocrine Pancreas ◽  
Specific Activity ◽  
Secretory Cells ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Secretory Product ◽  
Glycosyl Phosphatidylinositol ◽  
Granule Membrane ◽  
The One

Secretory proteins are segregated into two pathways out of the trans-Golgi network of regulated secretory cells. To identify proteins specifically secreted by pathways other than the one leading to zymogen granule exocytosis in the exocrine pancreas, conscious permanently cannulated pigs were perfused with atropine to inhibit the regulated fusion of granules. Atropine almost totally inhibited the protein secretion after 1 h of perfusion. The secretion of GP-2, a glycosyl phosphatidylinositol-anchored protein of the zymogen granule membrane, was partially inhibited but was never totally abolished by atropine perfusion. The pattern of proteins secreted under atropine was almost totally different. Soluble GP-2 was the major secretory product. Its specific activity increased 60 times over its normal level in all other conditions. This secretion clearly originated from nonregulated pathways. Results suggest that during the atropine block the apical plasmalemma could be the source of the released GP-2 and that the sustained nature of this release is compatible with a replenishment of the plasmalemma with GP-2 by the continuous exocytosis of vesicles from the nonregulated pathways.

scholarly journals Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Peixian Bai ◽  
Liyuan Wang ◽  
Kang Wei ◽  
Li Ruan ◽  
Liyun Wu ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Camellia Sinensis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Sequences ◽  
Biochemical Properties ◽  
High Similarity ◽  
New Gene ◽  
Tea Plants

Abstract Background Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. Results The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0–8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5′-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). Conclusions Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

scholarly journals Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase

1991 ◽  
Vol 266 (22) ◽  
pp. 14155-14158
Author(s):  
M.J. Berry ◽  
J.D. Kieffer ◽  
J.W. Harney ◽  
P.R. Larsen
Keyword(s):  
Biochemical Properties ◽  
Type I ◽  
Iodothyronine Deiodinase

scholarly journals SYNTHESIS AND MIGRATION OF PROTEINS IN THE CELLS OF THE EXOCRINE PANCREAS AS REVEALED BY SPECIFIC ACTIVITY DETERMINATION FROM RADIOAUTOGRAPHS

1963 ◽  
Vol 16 (1) ◽  
pp. 1-23 ◽  
Author(s):  
H. Warshawsky ◽  
C. P. Leblond ◽  
B. Droz
Keyword(s):  
Specific Activity ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
The Mean ◽  
And Migration ◽  
Rats And Mice ◽  
Silver Grains ◽  
Golgi Zone

Radioautographs of pancreatic acinar cells were prepared in rats and mice sacrificed at various times after injection of leucine-, glycine-, or methionine-H3. Measurements of radioactivity concentration (number of silver grains per unit area) and relative protein concentration (by microspectrophotometry of Millon-treated sections) yielded the mean specific activity of proteins in various regions of the acinar cells. The 2 to 5 minute radioautographs as well as the specific activity time curves demonstrate protein synthesis in ergastoplasm. From there, most newly synthesized proteins migrate to and accumulate in the Golgi zone. Then they spread to the whole zymogen region and, finally, enter the excretory ducts. An attempt at estimating turnover times indicated that two classes of proteins are synthesized in the ergastoplasm: "sedentary" with a slow turnover (62.5 hours) and "exportable" with rapid turnover (4.7 minutes). It is estimated that the exportable proteins spend approximately 11.7 minutes in the Golgi zone where they are built up into zymogen granules, and thereafter 36.0 minutes as fully formed zymogen granules, before they are released outside the acinar cell as pancreatic secretion. The mean life span of a zymogen granule in the cell is estimated to be 47.7 minutes.

scholarly journals ENHANCED ENZYMATIC ACTIVITY OF STREPTOMYCES GRISEOPLANUS L-ASPARGINASE VIA ITS INCORPORATION IN AN OIL-BASED NANOCARRIER

2020 ◽  
pp. 203-210
Author(s):  
ABEER A. EL-HADI ◽  
HANAN MOSTAFA AHMED ◽  
RANIA A. ZAKI ◽  
AMIRA MOHAMED MOHSEN
Keyword(s):  
Thermal Stability ◽  
Oleic Acid ◽  
Enzymatic Activity ◽  
Specific Activity ◽  
Residual Activity ◽  
Tween 80 ◽  
Biochemical Properties ◽  
Lymphocytic Leukemia ◽  
Tween 20 ◽  
Ph Range

Objective: L-asparaginase (L-asp) is a vital enzyme used as a therapeutic agent in combination with other drugs in the treatment of acute lymphoma, melanosarcoma and lymphocytic leukemia. Immobilization of enzymes through loading on nanoemulsion (NE) results in some advantages such as enhancing their stability and increasing their resistance to proteases. Aim of the present study is to formulate L-asp loaded nanoemulsion to enhance its efficiency and thermal stability. Methods: Nanoemulsion loaded with L-asp crude extract (specific activity 13.23U/mg protein) was prepared employing oleic acid as oil, tween 20/tween 80 as surfactants and propylene glycol (PG) as co-surfactant. L-asp loaded NE underwent several thermodynamic stability studies and the optimized formulae were further examined for their biochemical properties and thermal stability. Results The developed formulations were spherical in shape and their sizes were in the nanometric dimensions with negatively charged zeta potential values. Upon comparing the enzyme activity of L-asp loaded NE employing tween 20 (F1) or tween80 (F4) at different concentrations, the results revealed that F4 NE showed higher enzymatic activity [323 U/ml] compared to F1 NE [197 U/ml] at the same concentration. The nanosized immobilized L-asp was more stable in the pH range from 8 to 8.5 as compared to free L-asp. The immobilized enzyme preserved about 59.11% of its residual activity at 50 °C; while free L-asp preserved about 33.84%. Conclusion: In the view of these results, NE composed of oleic acid, tween 80 and PG represents a promising dosage form for enhancing the activity and stability of Streptomyces griseoplanus L-asp.

scholarly journals Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15.

2014 ◽  
Vol 61 (4) ◽  
Author(s):  
Mohd Adilin Yaacob ◽  
Wan Atiqah Najiah Wan Hasan ◽  
Mohd Shukuri Mohamad Ali ◽  
Raja Noor Zaliha Raja Abdul Rahman ◽  
Abu Bakar Salleh ◽  
...  
Keyword(s):  
Molecular Dynamic ◽  
Conformational Changes ◽  
Specific Activity ◽  
3D Structure ◽  
Coli Strain ◽  
Exchange Chromatography ◽  
Molecular Dynamic Simulations ◽  
Type I ◽  
Enzymatic Reactions ◽  
Dynamic Simulations

Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.

Purification and biochemical characterization of a β-cyanoalanine synthase expressed in germinating seeds of Sorghum bicolor (L.) moench

2018 ◽  
Vol 43 (6) ◽  
pp. 638-650
Author(s):  
Ruth Ololade Amiola ◽  
Adedeji Nelson Ademakinwa ◽  
Zainab Adenike Ayinla ◽  
Esther Nkechi Ezima ◽  
Femi Kayode Agboola
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Biochemical Properties ◽  
Subunit Molecular Weight ◽  
Cyanoalanine Synthase ◽  
Germinating Seeds ◽  
Sorghum Bicolor L

Abstract Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H2S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H2S/mL/min, 2.97×10−1 s−1 and 4.43×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H2S/mL/min, 10.71×10−1 s−1 and 4.06×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl2, EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.

Sign in / Sign up

Export Citation Format

Share Document