scholarly journals Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction?

2003 ◽  
Vol 23 (22) ◽  
pp. 8124-8136 ◽  
Author(s):  
Michael J. Seewald ◽  
Astrid Kraemer ◽  
Marian Farkasovsky ◽  
Carolin Körner ◽  
Alfred Wittinghofer ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Interaction Analysis ◽  
Catalytic Efficiency ◽  
Diffusion Control ◽  
Microtubule Organization ◽  
Rate Limiting Step ◽  
Association Reaction ◽  
Rate Limiting ◽  
Fold Stimulation

ABSTRACT RanBP type proteins have been reported to increase the catalytic efficiency of the RanGAP-mediated GTPase reaction on Ran. Since the structure of the Ran-RanBP1-RanGAP complex showed RanBP1 to be located away from the active site, we reinvestigated the reaction using fluorescence spectroscopy under pre-steady-state conditions. We can show that RanBP1 indeed does not influence the rate-limiting step of the reaction, which is the cleavage of GTP and/or the release of product Pi. It does, however, influence the dynamics of the Ran-RanGAP interaction, its most dramatic effect being the 20-fold stimulation of the already very fast association reaction such that it is under diffusion control (4.5 × 108 M−1 s−1). Having established a valuable kinetic system for the interaction analysis, we also found, in contrast to previous findings, that the highly conserved acidic C-terminal end of RanGAP is not required for the switch-off reaction. Rather, genetic experiments in Saccharomyces cerevisiae demonstrate a profound effect of the acidic tail on microtubule organization during mitosis. We propose that the acidic tail of RanGAP is required for a process during mitosis.

Heme Oxygenase-1/Carbon Monoxide: From Basic Science to Therapeutic Applications

2006 ◽  
Vol 86 (2) ◽  
pp. 583-650 ◽  
Author(s):  
Stefan W. Ryter ◽  
Jawed Alam ◽  
Augustine M. K. Choi
Keyword(s):  
Carbon Monoxide ◽  
Cellular Proliferation ◽  
Biochemical Characterization ◽  
Apoptotic Cell ◽  
Heme Oxygenase 1 ◽  
Bile Pigments ◽  
Cellular Functions ◽  
Rate Limiting Step ◽  
Rate Limiting

The heme oxygenases, which consist of constitutive and inducible isozymes (HO-1, HO-2), catalyze the rate-limiting step in the metabolic conversion of heme to the bile pigments (i.e., biliverdin and bilirubin) and thus constitute a major intracellular source of iron and carbon monoxide (CO). In recent years, endogenously produced CO has been shown to possess intriguing signaling properties affecting numerous critical cellular functions including but not limited to inflammation, cellular proliferation, and apoptotic cell death. The era of gaseous molecules in biomedical research and human diseases initiated with the discovery that the endothelial cell-derived relaxing factor was identical to the gaseous molecule nitric oxide (NO). The discovery that endogenously produced gaseous molecules such as NO and now CO can impart potent physiological and biological effector functions truly represented a paradigm shift and unraveled new avenues of intense investigations. This review covers the molecular and biochemical characterization of HOs, with a discussion on the mechanisms of signal transduction and gene regulation that mediate the induction of HO-1 by environmental stress. Furthermore, the current understanding of the functional significance of HO shall be discussed from the perspective of each of the metabolic by-products, with a special emphasis on CO. Finally, this presentation aspires to lay a foundation for potential future clinical applications of these systems.

scholarly journals Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis process

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Wenyan Ji ◽  
Wujin Sun ◽  
Jinmei Feng ◽  
Tianshun Song ◽  
Dalu Zhang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Acid Synthesis ◽  
Synthesis Process ◽  
Acetylneuraminic Acid ◽  
Cleavage Activity ◽  
Rate Limiting Step ◽  
Synthesis Activity ◽  
Rate Limiting ◽  
Class I Aldolase

Abstract N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the high Neu5Ac cleavage activity in most isozyme forms, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a “GRAS” (General recognized as safe) strain C. glutamicum ATCC 13032 (CgNal). Compared to all previously reported NALs, CgNal exhibited the lowest apparent k cat/Km value for Neu5Ac and highest apparent k cat/Km values for ManNAc and pyruvate, which makes CgNal favor industrial Neu5Ac synthesis process in a non-equilibrium condition. The recombinant CgNal reached the highest expression level (480 mg/L culture) and the highest reported yield of Neu5Ac was achieved (194 g/L, 0.63 M). All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition.

Molecular and biochemical characterization of Paragonimus westermani tyrosinase

Parasitology ◽  
2015 ◽  
Vol 142 (6) ◽  
pp. 807-815 ◽  
Author(s):  
Y.-A. BAE ◽  
S.-H. KIM ◽  
C.-S. AHN ◽  
J.-G. KIM ◽  
Y. KONG
Keyword(s):  
Oxidase Activity ◽  
Biochemical Characterization ◽  
Hydroxyl Groups ◽  
Reading Frame ◽  
Paragonimus Westermani ◽  
Rate Limiting Step ◽  
Tanning Process ◽  
Weak Activity ◽  
Rate Limiting

SUMMARYTrematode tyrosinases (TYRs) play a major role in the tanning process during eggshell formation. We investigated the molecular and biochemical features of Paragonimus westermani TYR (PwTYR). The PwTYR cDNA was composed of 1568-bp encompassing a 1422-bp-long open reading frame (474-amino acid polypeptide). A strong phylogenetic relationship with Platyhelminthes and Deuterostomian orthologues was evident. The recombinant PwTYR expressed in prokaryotic cells promptly oxidized diphenol substrates, with a preferential affinity toward ortho-positioned hydroxyl groups. It demonstrated fairly weak activity for monophenol compounds. Diphenol oxidase activity was augmented with an increase of pH from 5·0 to 8·0, while monophenol oxidase activity was highest at an acidic pH and gradually decreased as pH increased. Transcription profile of PwTYR was temporally upregulated along with worm development. PwTYR was specifically localized in vitellocytes and eggs. The results suggested that conversion of tyrosine to L-dihydroxyphenylalanine by PwTYR monophenol oxidase activity might be rate-limiting step during the sclerotization process of P. westermani eggs. The pH-dependent pattern of monophenol and diphenol oxidase activity further proposes that the initial hydroxylation might slowly but steadily progress in acidic secreted vesicles of vitellocytes and the second oxidation process might be rapidly accelerated by neural or weak alkaline pH environments within the ootype.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

Wavelet Based Structural Analysis of Electroless Deposits in the Diffusion Limited Regime

1994 ◽  
Vol 367 ◽  
Author(s):  
A. Arneodo ◽  
F. Argoul ◽  
A. Kuhn ◽  
J.F. Muzy
Keyword(s):  
Electroless Deposition ◽  
Side Reactions ◽  
Statistical Features ◽  
Diffusion Limited ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Fractal Patterns ◽  
Rate Limiting ◽  
Comparative Structural Characterization

AbstractWe discuss the actual relevance of thin gap geometry electrodeposition to generate fractal patterns that mimic the morphology of Witten and Sander's diffusion-limited aggregates (DLA). Eliminating migration and convection, as well as electrochemical side reactions, we show that electroless deposition is a good candidate to meet the requirements for diffusion to be the rate limiting step of the growth process. We use the wavelet transform microscope to achieve a comparative structural characterization of both experimental electroless deposits and numerical DLA clusters. The fact that five-fold symmetry and Fibonacci hierarchical ordering are found as common predominant statistical features is, to our knowledge, the first demonstration, relying on an appropriate structural fractal analysis, of the existence of DLA morphologies in an experimental context.

scholarly journals Comparative Biochemical Characterization of Three Exolytic Oligoalginate Lyases from Vibrio splendidus Reveals Complementary Substrate Scope, Temperature, and pH Adaptations

2014 ◽  
Vol 80 (14) ◽  
pp. 4207-4214 ◽  
Author(s):  
Sujit Sadashiv Jagtap ◽  
Jan-Hendrik Hehemann ◽  
Martin F. Polz ◽  
Jung-Kul Lee ◽  
Huimin Zhao
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Vibrio Splendidus ◽  
Marine Microbes ◽  
Ph Optimum ◽  
Brown Seaweeds ◽  
Content Type ◽  
Alginate Lyases

ABSTRACTMarine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exo-type oligoalginate lyases (OalA, OalB, and OalC) fromVibrio splendidus12B01 (12B01), a marine bacterioplankton species. OalA was most active at 16°C, had a pH optimum of 6.5, and displayed activities toward poly-β-d-mannuronate [poly(M)] and poly-α-l-guluronate [poly(G)], indicating that it is a bifunctional enzyme. OalB and OalC were most active at 30 and 35°C, had pH optima of 7.0 and 7.5, and degraded poly(M·G) and poly(M), respectively. Detailed kinetic analyses of oligoalginate lyases with poly(G), poly(M), and poly(M·G) and sodium alginate as substrates demonstrated that OalA and OalC preferred poly(M), whereas OalB preferred poly(M·G). The catalytic efficiency (kcat/Km) of OalA against poly(M) increased with decreasing size of the substrate. OalA showedkcat/Kmfrom 2,130 mg−1ml s−1for the trisaccharide to 224 mg−1ml s−1for larger oligomers of ∼50 residues, and 50.5 mg−1ml s−1for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.

Vapor Transport Deposition and Characterization of Polycrystalline CdTe Solar Absorbers

2003 ◽  
Vol 763 ◽  
Author(s):  
James M. Kestner ◽  
Sarah McElvain ◽  
Colin A. Wolden ◽  
Stephen Kelly ◽  
Tim R. Ohno ◽  
...  
Keyword(s):  
Vapor Transport ◽  
Buffer Layers ◽  
High Rate ◽  
X Ray Diffraction ◽  
Solar Absorbers ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Substrate Temperatures ◽  
Vapor Transport Deposition

AbstractVapor transport deposition is being developed for high-rate synthesis of CdTe thin films. Films have been deposited at rates in excess of 20 μm/min. Thegrowth ratedependenceon source temperature yielded an apparent activation energy of 42 kcal/mol, in good agreement with the theoretical value for CdTe sublimation (45.7 kcal/mol). For substrate temperatures greater than 400°C the rate limiting step was resublimation. This phenomenon had a dramatic influence on morphology, although x-ray diffraction of all films indicated a strong (111) orientation. A preliminary device optimization investigating the effect of CdTe deposition temperature, post-deposition CdCl2 anneal parameters, alternative back contacts, and high-resistance buffer layers yielded a best cell with efficiency of 9.8% (704 mV Voc, 21.0mA/cm2 Jsc, 66% FF).

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

2013 ◽  
Vol 8 (12) ◽  
pp. 1183-1193 ◽  
Author(s):  
Marcin Maciąga ◽  
Michał Szkop ◽  
Andrzej Paszkowski
Keyword(s):  
Common Wheat ◽  
Aspartate Aminotransferase ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Amino Acid Sequences ◽  
Molecular Weights ◽  
Sds Page ◽  
Technique Comparison

AbstractSix allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k cat) and the k cat/K m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s−1/10,412–10,795 s−1 M−1 for AAT-2 zone and 4.6–7.0 s−1/527–700 s−1 M−1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.

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