scholarly journals Probing the active site of cytoplasmic aldehyde dehydrogenase with a chromophoric reporter group

1994 ◽  
Vol 300 (1) ◽  
pp. 25-30 ◽  
Author(s):  
T M Kitson ◽  
K E Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Negative Charge ◽  
Positive Charge ◽  
Gel Filtration ◽  
Modified Enzyme ◽  
Covalently Linked ◽  
Reporter Group

3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one (‘DMNB’) reacts with cytoplasmic aldehyde dehydrogenase in a similar way to that previously observed with the structurally related p-nitrophenyl dimethylcarbamate, but provides a covalently linked p-nitrophenol-containing reporter group at the enzyme's active site. The pKa of the enzyme-linked reporter group is much higher than that of free p-nitrophenol, which is consistent with its being in a very hydrophobic environment, or possibly one containing negative charge. Upon binding of NAD+ to the modified enzyme, the pKa falls dramatically, by about 4 1/2 pH units. This implies that under these conditions there is a positive charge near the p-nitrophenoxide moiety, perhaps that of the nicotinamide ring of NAD+. The modified enzyme binds NAD+ very tightly; neither gel filtration nor dialysis is effective in separating them. However, the reporter group provides a convenient way of monitoring the displacement of this bound NAD+ when NADH is added.

scholarly journals Purification and Electrophoretic Behavior of Sugarcane Invertases

1969 ◽  
Vol 51 (1) ◽  
pp. 39-45
Author(s):  
Alex G. Alexander
Keyword(s):  
Enzyme Purification ◽  
Negative Charge ◽  
Positive Charge ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Good Separation ◽  
Electrophoretic Behavior ◽  
Negative Electrodes ◽  
Differential Solubility ◽  
Fair Degree

A fair degree of sugarcane invertase purification has been achieved by techniques of differential solubility, gel filtration, and paper electrophoresis. Invertase is readily salted out with ammoniun sulfate between 38- and 52- percent saturation. Activity is largely lost during dialysis against distilled water, but is regained by passage through columns of G-200 Sephadex gel. Reactivation is attributed to removal of unknown inhibitors. Filtration did not accomplish good separation of invertase from other protein. Electrophoresis experiments showed that invertases are quite mobile compared to contaminant protein, and move quickly toward positive and negative electrodes. Two distinct invertase areas were obtained free of contaminant protein, one enzyme bearing a positive charge and the other a negative charge. Filtration and electrophoresis steps described herein achieved good enzyme purification without use of glucose or manganese, and thus avoided the appearance of reconstituted invertase.

Interaction of monomeric and polymeric species of metal ions with clay surfaces. III. Aluminium(III) and chromium(III)

Soil Research ◽  
1978 ◽  
Vol 16 (1) ◽  
pp. 53 ◽  
Author(s):  
P Rengasamy ◽  
JM Oades
Keyword(s):  
Molecular Weight ◽  
Metal Ion ◽  
Negative Charge ◽  
Positive Charge ◽  
Gel Filtration ◽  
Charge Reversal ◽  
Original Solution ◽  
Polymeric Species ◽  
Surface Hydroxyls ◽  
Nitrate Solutions

Hydrolysis and polymerization in aluminium nitrate and chromic nitrate solutions with different metal/OH ratios were studied by gel filtration chromatography and ultrafiltration techniques. The characteristics of poly[Al(III)-OH] cations separated by ultrafiltration depended on the molecular weight which was controlled by the OH/Al ratio of the original solution. When the OH/Al ratio was <2.0, the polycations had molecular weight < 50 000, high positive charge and were stable. When the ratio was > 2.0 , the polycations had molecular weight > 100 000, low positive charge and rapidly condensed to gibbsite. Polymerization in chromic nitrate solutions was slow and the separated poly[Cr(III)-OH] cations had low molecular weight (<20000), high positive charge and were stable. The interaction of monomeric and polymeric species of aluminium(III) and chromium(III) with the surfaces of sodium-kaolinite, sodium-bentonite and sodium-illite was studied using hydrolysed nitrate solutions of aluminium and chromium, and separated polycations. The results obtained were consistent with the earlier observations of the iron(III) system, and the behaviour could be explained on the basis of positive charge and probable structure of polycations. The adsorption of aluminium(III) and chromium(III) on clay surfaces from hydrolysed metal ion solutions was related to OH/Al or OH/Cr ratios and pH. The adsorption of polycations was low and related to their positive charge. Maximum adsorption resulted in charge reversal on clays. Addition of the hydrolysed nitrate solutions to clays also caused charge reversal and reduction of negative charge. The adsorption from a mixture of monomers and polymers on bentonite surfaces reduced negative charge markedly due to interlayer formation, while the adsorption of separated polycations in low amounts neutralized the charge on surface hydroxyls and led to the flocculation of clays. The anomalous behaviour of the poly[Al(III)-OH] cations of molecular weight >100 000 could be explained if they consisted of planar sheets, formed from coalesced rings of aluminium octahedra, which crystallized into gibbsite.

scholarly journals Structural environment of an essential cysteine residue of xylanase from Chainia sp. (NCL 82.5.1)*

1996 ◽  
Vol 316 (3) ◽  
pp. 771-775 ◽  
Author(s):  
Mala RAO ◽  
Suvarna KHADILKAR ◽  
Kavita R. BANDIVADEKAR ◽  
Vasanti DESHPANDE
Keyword(s):  
Active Site ◽  
Gel Filtration ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Cysteine Residue ◽  
Yellow Colour ◽  
Streptomyces Species ◽  
Complete Inactivation ◽  
Structural Environment ◽  
Reporter Group

N-(2,4-Dinitroanilino)maleimide (DAM) reacts covalently with the thiol group of the xylanase from Chainia leading to complete inactivation in a manner similar to N-ethylmaleimide, but provides a reporter group at the active site of the enzyme. Increasing amounts of xylan offered enhanced protection against inactivation of the xylanase by DAM. Xylan (5 mg) showed complete protection, providing evidence for the presence of cysteine at the substrate-binding site of the enzyme. Kinetics of chemical modification of the xylanase by DAM indicated the involvement of l mol of cysteine residue per mol of enzyme, as reported earlier [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172–177]. The second-order rate constant for the reaction of DAM with the enzyme was 3.61×103 M-1·min-1. The purified xylanase was alkylated with DAM and digested with pepsin. The peptides were separated by gel filtration. The specific modified cysteinyl peptide was further purified by reverse-phase HPLC. The active-site peptide was located visually by its predominant yellow colour and characterized by a higher A340 to A210 ratio. The modified active-site peptide has the sequence: Glu-Thr-Phe-Xaa-Asp. The sequence of the peptide was distinctly different from that of cysteinyl peptide derived from a xylanase from a thermotolerant Streptomyces species, but showed the presence of a conserved aspartic acid residue consistent with the catalytic regions of other glucanases.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

scholarly journals HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fa-Hui Sun ◽  
Peng Zhao ◽  
Nan Zhang ◽  
Lu-Lu Kong ◽  
Catherine C. L. Wong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Active Site ◽  
Negative Charge ◽  
Structural Data ◽  
Dna Breaks ◽  
Adp Ribosylation ◽  
Local Conformation ◽  
Key Residues ◽  
Structural Insights

AbstractUpon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

scholarly journals Zerumbone Inhibits Helicobacter pylori Urease Activity

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2663
Author(s):  
Hyun Jun Woo ◽  
Ji Yeong Yang ◽  
Pyeongjae Lee ◽  
Jong-Bae Kim ◽  
Sa-Hyun Kim
Keyword(s):  
Helicobacter Pylori ◽  
Carbon Atom ◽  
Natural Compound ◽  
Urease Activity ◽  
Negative Charge ◽  
Positive Charge ◽  
Gastric Epithelium ◽  
Functional Inhibition ◽  
Electrical Gradient ◽  
H Pylori

Helicobacter pylori (H. pylori) produces urease in order to improve its settlement and growth in the human gastric epithelium. Urease inhibitors likely represent potentially powerful therapeutics for treating H. pylori; however, their instability and toxicity have proven problematic in human clinical trials. In this study, we investigate the ability of a natural compound extracted from Zingiber zerumbet Smith, zerumbone, to inhibit the urease activity of H. pylori by formation of urease dimers, trimers, or tetramers. As an oxygen atom possesses stronger electronegativity than the first carbon atom bonded to it, in the zerumbone structure, the neighboring second carbon atom shows a relatively negative charge (δ−) and the next carbon atom shows a positive charge (δ+), sequentially. Due to this electrical gradient, it is possible that H. pylori urease with its negative charges (such as thiol radicals) might bind to the β-position carbon of zerumbone. Our results show that zerumbone dimerized, trimerized, or tetramerized with both H. pylori urease A and urease B molecules, and that this formation of complex inhibited H. pylori urease activity. Although zerumbone did not affect either gene transcription or the protein expression of urease A and urease B, our study demonstrated that zerumbone could effectively dimerize with both urease molecules and caused significant functional inhibition of urease activity. In short, our findings suggest that zerumbone may be an effective H. pylori urease inhibitor that may be suitable for therapeutic use in humans.

scholarly journals Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology

1979 ◽  
Vol 183 (3) ◽  
pp. 615-622 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Sequence Homology ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Terminal Sequence ◽  
Complement Components ◽  
Factor B ◽  
Terminal Residue ◽  
Covalently Linked

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

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