scholarly journals An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method

RNA ◽  
2008 ◽  
Vol 14 (5) ◽  
pp. 938-949 ◽  
Author(s):  
D. Jaikaran ◽  
M. D. Smith ◽  
R. Mehdizadeh ◽  
J. Olive ◽  
R. A. Collins
Keyword(s):  
Cleavage Reaction ◽  
Vs Ribozyme ◽  
Nucleotide Analog ◽  
Incorporation Method

FACTOR XIII AND ITS NATURAL PLASMATIC TARGETS:FIBRINO-GEN,FIBRONECTIN AND ALPHA2-ANTIPLASMIN IN MALIGNANT DISEASE

1987 ◽  
Author(s):  
J Kioczko ◽  
M Wojtukiewicz ◽  
M Bielawiec ◽  
E Pilecka
Keyword(s):  
Malignant Disease ◽  
Plasma Concentrations ◽  
Subunit A ◽  
Lysis Time ◽  
At Iii ◽  
Euglobulin Lysis Time ◽  
Monospecific Antisera ◽  
Melanoma Malignum ◽  
Incorporation Method

The relevance of fibrin formation and its stabilization in tumour growth and metastasis formation seems to be important but it still remains unclear.The role of F.XIII in this process may emerge from a variety of mechanisms it is involved in,including crosslink of fibrin molecules,α2AP incorporation into fibrin and reciprocal crosslink of fibronectin,fibrin and collagen. We studied F.XIII and its natural targets such as fibrinogen , fibronectin and α2AP in 61 patients with various inoperable neoplasms (21 with Ca mammae,18 with Ca ventriculi and 22 with Melanoma malignum).F.XIII activity was measured by dansylcadaverine incorporation method acc.to Lorand et α2F.XIII subunit “a” and “b”, f ibronect in , α2AP , AT III, α22M , α2AT and C-I plasma concentrations were estimated by means of rocket immunoelectrophoresis acc.to Laurell using monospecific antisera (Behringwerke AG,Marburg).The following tests were performed in all cases:fibrinogen concentration, euglobulin lysis time and serum FDP.In comparison with healthy subjects the patients revealed statistically significant decrease in F.XIII activity and its subunit “a“ and “b“ concentrations concomitant with lowered fibronectin and α2AP levels.No statistically significant correlations were found between F.XIII and its natural plasmatic substrates.Furthermore,the malignant patients showed decreased AT III concentrations,whereas α2CiI and α2AT levels were elevated.Prolongation of ELT concomitant with an increase of FDP concentration were also found. Differences in F.XIII level and in other haemostasis parameters were stated between different tumour types. Our data indicate that the role of F.XIII in malignancy is not limited to fibrin stabilization but its interactions with fibronectin and α2AP should be taken into account.

Role of Nd3+and Pb2+on the RNA Cleavage Reaction by a Small Ribozyme†

Biochemistry ◽  
1997 ◽  
Vol 36 (12) ◽  
pp. 3514-3521 ◽  
Author(s):  
Tatsuo Ohmichi ◽  
Naoki Sugimoto
Keyword(s):  
Rna Cleavage ◽  
Cleavage Reaction

Role of divalent metal ions in the hammerhead RNA cleavage reaction

Biochemistry ◽  
1991 ◽  
Vol 30 (39) ◽  
pp. 9464-9469 ◽  
Author(s):  
SueAnn C. Dahm ◽  
Olke C. Uhlenbeck
Keyword(s):  
Metal Ions ◽  
Divalent Metal ◽  
Rna Cleavage ◽  
Divalent Metal Ions ◽  
Cleavage Reaction ◽  
Hammerhead Rna

scholarly journals The role of an active site Mg2+ in HDV ribozyme self-cleavage: insights from QM/MM calculations

2015 ◽  
Vol 17 (1) ◽  
pp. 670-679 ◽  
Author(s):  
Vojtěch Mlýnský ◽  
Nils G. Walter ◽  
Jiří Šponer ◽  
Michal Otyepka ◽  
Pavel Banáš
Keyword(s):  
Active Site ◽  
The Self ◽  
Direct Impact ◽  
Cleavage Reaction ◽  
Hdv Ribozyme ◽  
Specific Position

The specific position and coordination of active site Mg2+ ion have a significant direct impact on the self-cleavage reaction in HDV ribozyme.

Fes1p acts as a nucleotide exchange factor for the ribosome-associated molecular chaperone Ssb1p

2006 ◽  
Vol 387 (12) ◽  
pp. 1593-1600 ◽  
Author(s):  
Zdravko Dragovic ◽  
Yasuhito Shomura ◽  
Nikolay Tzvetkov ◽  
F. Ulrich Hartl ◽  
Andreas Bracher
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Chaperone ◽  
Point Mutations ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Analog ◽  
Nucleotide Exchange Factor ◽  
Similar Affinity ◽  
Stimulation Of

Abstract The HspBP1 homolog Fes1p was recently identified as a nucleotide exchange factor (NEF) of Ssa1p, a canonical Hsp70 molecular chaperone in the cytosol of Saccharomyces cerevisiae. Besides the Ssa-type Hsp70s, the yeast cytosol contains three additional classes of Hsp70, termed Ssb, Sse and Ssz. Here, we show that Fes1p also functions as NEF for the ribosome-bound Ssb Hsp70s. Sequence analysis indicated that residues important for interaction with Fes1p are highly conserved in Ssa1p and Ssb1p, but not in Sse1p and Ssz1p. Indeed, Fes1p interacts with Ssa1p and Ssb1p with similar affinity, but does not form a complex with Sse1p. Functional analysis showed that Fes1p accelerates the release of the nucleotide analog MABA-ADP from Ssb1p by a factor of 35. In contrast to the interaction between mammalian HspBP1 and Hsp70, however, addition of ATP only moderately decreases the affinity of Fes1p for Ssb1p. Point mutations in Fes1p abolishing complex formation with Ssa1p also prevent the interaction with Ssb1p. The ATPase activity of Ssb1p is stimulated by the ribosome-associated complex of Zuotin and Ssz1p (RAC). Interestingly, Fes1p inhibits the stimulation of Ssb1p ATPase by RAC, suggesting a complex regulatory role of Fes1p in modulating the function of Ssb Hsp70s in co-translational protein folding.

scholarly journals An analysis of the role of active site protic residues of cytochrome P-450s: mechanistic and mutational studies on 17α-hydroxylase-17,20-lyase (P-45017α also CYP17)

1998 ◽  
Vol 330 (2) ◽  
pp. 967-974 ◽  
Author(s):  
Peter LEE-ROBICHAUD ◽  
E. Monika AKHTAR ◽  
Muhammad AKHTAR
Keyword(s):  
Hydrogen Bonds ◽  
Active Site ◽  
Catalytic Properties ◽  
Active Form ◽  
Proton Donor ◽  
Wild Type ◽  
Cleavage Reaction ◽  
Acyl Carbon ◽  
Hydroxylation Reaction

Certain cytochrome P-450s involved in the transformation of steroids catalyse not only the hydroxylation process associated with the group of enzymes, but also an acyl-carbon cleavage reaction. The hydroxylation occurs using an iron-monooxygen species while the acyl-carbon cleavage has been suggested to be promoted by an iron peroxide. In this paper we have studied the role of active site protic residues, Glu305 and Thr306, in modulating the two activities. For this purpose, the kinetic parameters for the hydroxylation reaction (pregnenolone → 17α-hydroxypregnenolone) and two different versions of acyl-carbon cleavage (17α-hydroxypregnenolone → dehydroepiandrosterone and 3β-hydroxyandrost-5-ene-17β-carbaldehyde → 3β-hydroxyandrost-5,16-diene+androst-5-ene-3β,17α-diol) were determined using the wild-type human CYP17 and its eight different single and double mutants. In addition the propensity of the proteins to undergo a subtle rearrangement converting the 450 nm active-form into an inactive counterpart absorbing at 420 nm, was monitored by measuring the of the P-450 → P-420 conversion. The results are interpreted to draw the following conclusions. The functional groups of Glu305 and Thr306 do not directly participate in the two proton delivery steps required for hydroxylation but may be important participants for the provision of a net work of hydrogen bonds for ‘activating’ water that then acts as a proton donor. The loss of any one of these residues is, therefore, only partially debilitating. That the mutation of Thr306 impairs the hydroxylation reaction more than it does the acyl-carbon cleavage is consistent with the detailed mechanistic scheme considered in this paper. Furthermore attention is drawn to the fact that the mutation of Glu305 and Thr306 subtly perturbed the architecture of the active site, which affects the geometry of this region of the protein and therefore its catalytic properties.

scholarly journals Mutational Scan of the Human Immunodeficiency Virus Type 2 Integrase Protein

1998 ◽  
Vol 72 (5) ◽  
pp. 3916-3924 ◽  
Author(s):  
Fusinita M. I. van den Ent ◽  
Arnold Vos ◽  
Ronald H. A. Plasterk
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Cleavage Reaction ◽  
Viral Dna ◽  
Dna Integration ◽  
Immunodeficiency Virus

ABSTRACT Retroviral integrase (IN) cleaves linear viral DNA specifically near the ends of the DNA (cleavage reaction) and subsequently couples the processed ends to phosphates in the target DNA (integration reaction). In vitro, IN catalyzes the disintegration reaction, which is the reverse of the integration reaction. Ideally, we would like to test the role of each amino acid in the IN protein. We mutagenized human immunodeficiency virus type 2 IN in a random way using PCR mutagenesis and generated a set of mutants in which 35% of all residues were substituted. Mutant proteins were tested for in vitro activity, e.g., site-specific cleavage of viral DNA, integration, and disintegration. Changes in 61 of the 90 proteins investigated showed no phenotypic effect. Substitutions that changed the choice of nucleophile in the cleavage reaction were found. These clustered around the active-site residues Asp-116 and Glu-152. We also found alterations of amino acids that affected cleavage and integration differentially. In addition, we analyzed the disintegration activity of the proteins and found substitutions of amino acids close to the dimer interface that enhanced intermolecular disintegration activity, whereas other catalytic activities were present at wild-type levels. This study shows the feasibility of investigating the role of virtually any amino acid in a protein the size of IN.

scholarly journals Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

1998 ◽  
Vol 180 (17) ◽  
pp. 4658-4666 ◽  
Author(s):  
Mary McLenigan ◽  
Thomas S. Peat ◽  
Ekaterina G. Frank ◽  
John P. McDonald ◽  
Martín Gonzalez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Potential Effect ◽  
Wild Type ◽  
Cleavage Reaction ◽  
Nonsense Mutations ◽  
Spontaneous Mutagenesis ◽  
Sos Mutagenesis ◽  
Low Copy Number ◽  
Induced Mutagenesis

ABSTRACT Although it has been 10 years since the discovery that theEscherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomalumuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′.

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