scholarly journals Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica

1998 ◽  
Vol 329 (3) ◽  
pp. 659-664 ◽  
Author(s):  
Zhihong DENG ◽  
Min HUANG ◽  
Kuber SINGH ◽  
A. Richard ALBACH ◽  
P. Steven LATSHAW ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Specific Activity ◽  
Ph Dependence ◽  
Cloning And Expression ◽  
Kinetic Properties ◽  
Partial Amino Acid Sequence ◽  
Sequence Identity ◽  
Cloned Cdna ◽  
Cloned Gene ◽  
Substrate Affinities

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites. Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme. The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E. histolytica [Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim. Biophys. Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem. J. 316, 57-63]. The sequence identity between the two proteins was 17%. The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs. The cloned cDNA was expressed and the protein purified. The kinetic properties were identical with those of the enzyme isolated from the organism. Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E. histolytica PFK gene [Bruchhaus et al. (1996)]. The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E. histolytica trophozoites.

scholarly journals Purification, cDNA cloning and expression of 15-oxoprostaglandin 13-reductase from pig lung

1998 ◽  
Vol 330 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Charles Mark ENSOR ◽  
Hongxing ZHANG ◽  
Hsin-Hsiung TAI
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Leukotriene B4 ◽  
Cloning And Expression ◽  
Library Screening ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass ◽  
Apparent Homogeneity ◽  
Pcr Product ◽  
Cloned Cdna

15-Oxoprostaglandin 13-reductase (PGR) has been purified to apparent homogeneity from pig lung. The enzyme was estimated to have a molecular mass of 36 kDa by both SDS/PAGE and non-denaturing PAGE, indicating that the enzyme is a monomer. 15-Oxo-PGE1, 15-oxo-PGE2 and 15-oxo-PGF2α were found to be substrates for the enzyme, whereas the corresponding 15-hydroxyprostaglandins were not. The reverse reaction, the oxidation of 13,14-dihydro-15-oxo-PGE1 to 15-oxo-PGE1, was not observed. Either NADH or NADPH could serve as a coenzyme. However, the Vmax with NADH was approx. 3-fold that with NADPH, while the Km for NADPH was approx. one-tenth that for NADH. Cloning of the cDNA was achieved by PCR and library screening. A 600 bp PCR product containing the sequences of three different tryptic peptides derived from purified PGR was used for cDNA library screening by plaque hybridization. A cDNA clone that contained the entire PGR coding sequence of 987 bp was obtained. The sequence codes for a protein of 329 amino acid residues with a calculated molecular mass of 35791 Da. Homology analysis indicated that the sequence is virtually identical with that of leukotriene B4 (LTB4) 12-hydroxydehydrogenase [Yokomizo, Ogawa, Uozumi, Kume, Izumi and Shimizu (1996) J. Biol. Chem. 271, 2844-2850]. Expression of this cDNA in Escherichia coli resulted in a protein exhibiting both PGR and LTB4 12-hydroxydehydrogenase activities. However, the specific activity of PGR with 15-oxo-PGE1 as a substrate was approx. 300-fold that of LTB4 12-hydroxydehydrogenase. These results indicate that the cloned cDNA codes for a protein with two different enzyme activities, with 15-oxoprostaglandins as the preferred substrates.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

Large-scale production of citrate synthase from a cloned gene

1982 ◽  
Vol 60 (12) ◽  
pp. 1143-1147 ◽  
Author(s):  
Harry W. Duckworth ◽  
Alexander W. Bell
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Tetracycline Resistance ◽  
Scale Production ◽  
Ampicillin Resistance ◽  
Colicin E1 ◽  
Large Scale Production ◽  
Cloned Gene

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

scholarly journals Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages.

1995 ◽  
Vol 182 (5) ◽  
pp. 1423-1433 ◽  
Author(s):  
A Campos-Neto ◽  
L Soong ◽  
J L Cordova ◽  
D Sant'Angelo ◽  
Y A Skeiky ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Leishmania Donovani ◽  
Class Ii ◽  
Cloning And Expression ◽  
Diagnostic Tools ◽  
Reading Frame ◽  
Dna Fragment ◽  
Peptide Gene ◽  
Cloned Gene ◽  
Sense Primer

The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen a L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-gamma and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasite interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.

pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli

Biochemistry ◽  
1991 ◽  
Vol 30 (23) ◽  
pp. 5750-5754 ◽  
Author(s):  
Dominique Deville-Bonne ◽  
Florence Bourgain ◽  
Jean Renaud Garel
Keyword(s):  
Escherichia Coli ◽  
Ph Dependence ◽  
Kinetic Properties

Purification to homogeneity and characterization of nonproteolyzed potato (Solanum tuberosum) tuber hexokinase 1

Botany ◽  
2011 ◽  
Vol 89 (5) ◽  
pp. 289-299 ◽  
Author(s):  
Marie-Claude Moisan ◽  
Jean Rivoal
Keyword(s):  
Solanum Tuberosum ◽  
Specific Activity ◽  
Solanum Tuberosum L ◽  
Extraction Procedure ◽  
Hydrophobic Interaction Chromatography ◽  
Solanum Chacoense ◽  
Kinetic Properties ◽  
Chromatographic Separations ◽  
The Stability ◽  
Fast Flow

We have developed an extraction procedure that improves the stability of potato ( Solanum tuberosum L.) tuber hexokinase (HK) after extraction. Using this protocol, we showed that at least four HK isoforms are present in this tissue, and they can be separated by hydrophobic-interaction chromatography on a butyl-Sepharose™ 4 Fast Flow column. One of the main HK isoforms was purified to homogeneity using further chromatographic separations on red dye, DEAE Fractogel, hydroxyapatite, cibacron blue, and MonoQ matrices. HK-specific activity of this fraction (10.2 U·mg protein–1) corresponds to an enrichment of more than 5500-fold, with a yield of 0.9%. This is the highest reported HK-specific activity from a plant source. The purified enzyme consisted of a monomer with a subunit apparent Mr of 51 kDa when analyzed by SDS–PAGE. This polypeptide was recognized by affinity-purified anti- Solanum chacoense Bitt. recombinant HK IgGs. The protein was digested with trypsin and its digestion products were subjected to MS – MS sequencing after HPLC separation. The sequences of these tryptic peptides matched the predicted coding sequence of the S. tuberosum HK1 gene with a coverage of 57%. Examination of the kinetic properties of the purified protein HK1 indicates that it may be regulated by the internal O2 concentration of the tuber because of its sensitivity to acidic pHs and inhibition by ADP.

scholarly journals Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

2018 ◽  
Vol 22 (2) ◽  
pp. 55
Author(s):  
Enny Ratnaningsih ◽  
Idris Idris
Keyword(s):  
Recombinant Protein ◽  
Bacillus Cereus ◽  
Tertiary Structure ◽  
Specific Activity ◽  
Halogen Bond ◽  
Expression System ◽  
Industrial Sector ◽  
Cloning And Expression ◽  
Haloacid Dehalogenase ◽  
Organohalogen Compounds

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0  using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily.

Mechanism of activation of renal Na+-K+-ATPase in the rat: effects of potassium loading

1980 ◽  
Vol 238 (4) ◽  
pp. F315-F323 ◽  
Author(s):  
H. J. Rodriguez ◽  
W. C. Hogan ◽  
R. N. Hellman ◽  
S. Klahr
Keyword(s):  
Thyroid Hormones ◽  
Kinetic Parameters ◽  
Enzyme Induction ◽  
Specific Activity ◽  
Rat Kidney ◽  
Enzymatic Reaction ◽  
Kinetic Properties ◽  
Renal Growth ◽  
Hill Coefficients ◽  
Potassium Loading

The mechanism of activation of Na+-K+-ATPase after chronic potassium loading has been investigated in the rat kidney. Potassium loading stimulated the specific activity of Na+-K+-ATPase in the cortex and medulla of the kidney. This effect was not accompanied by a generalized increase in the cellular contents of RNA and proteins and could not be accounted for by an effect of potassium loading on renal growth. Enzyme induction does not appear to be mediated by changes in the endogenous levels of glucocorticoid or thyroid hormones. Evidence obtained from investigation of the partial reactions (Pi intermediate, ouabain-sensitive pNPPase) of the Na+-K+-ATPase enzymatic reaction is consistent with the interpretation that chronic potassium loading in the rat increases the number of enzyme units (Na+ pumps) in the cortex of the kidney. Analysis of the kinetic parameters (Km, K1/2, Vmax, Hill coefficients) of the enzymatic reaction indicates that K+ loading has little or no effect on the kinetic properties (affinity, cooperativity) of the stimulated transport enzyme.

scholarly journals Changes in Activity and Kinetic Properties of the Proteasome in Different Rat Organs during Development and Maturation

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
A. Petersen ◽  
A. Honarvar ◽  
M. Zetterberg
Keyword(s):  
Rat Brain ◽  
Structural Changes ◽  
Specific Activity ◽  
Kinetic Properties ◽  
Loss Of Function ◽  
Proteolytic System ◽  
Peptide Substrates ◽  
Menten Constant ◽  
Fluorogenic Peptide ◽  
Soluble Fractions

The proteasome is considered the most important proteolytic system for removal of damaged proteins with aging. Using fluorogenic peptide substrates, the chymotrypsin-like, the trypsin-like, and the peptidylglutamyl peptidase activities of the proteasome were measured in the soluble fractions of liver, brain, and lens rat homogenates. Specific activity was significantly decreased in liver and brain homogenates with maturation of the animal, that is, from newborn (7 days old) to fertile rats (2–4 months old). Rat lens homogenate exhibited an increase in activity with maturation and also with aging. Chymotrypsin-like activity was stimulated by calcium and this proteolytic activity was significantly decreased with maturation of the rat brain. The Michaelis-Menten constant (Km) increased with age in rat liver and lens, indicating a loss of affinity for its substrates by the proteasome in the animal with maturation and aging. The present data suggest that the loss of function of the proteasome with maturation may be due to structural changes of the proteasome or a decreased content of regulatory components.

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