Cloning, functional expression, and complementation analysis of an inorganic pyrophosphatase fromBartonella bacilliformis

1997 ◽  
Vol 43 (8) ◽  
pp. 734-743 ◽  
Author(s):  
Samuel J. Mitchell ◽  
Michael F. Minnick
Keyword(s):  
Amino Acid ◽  
Functional Expression ◽  
Relative Activity ◽  
Inorganic Pyrophosphatase ◽  
Amino Acid Residues ◽  
Bartonella Bacilliformis ◽  
E Coli ◽  
Catalytic Active Site

We have cloned the inorganic pyrophosphatase gene (ppa) from the facultative intracellular pathogen Bartonella bacilliformis and characterized its encoded product. The 531-bp gene is located approximately 1 kb downstream of, and in opposite orientation to, the invasion-associated locus (ialAB) of B. bacilliformis. The predicted protein encoded by ppa is 177 amino acid residues, which is in agreement with in vitro and in vivo synthesis of a protein with an apparent molecular mass of 22–23 kDa. The predicted B. bacilliformis pyrophosphatase (PPase) sequence is 53% identical and 85% similar to the E. coli PPase (EC 3.6.1.1), and contains all 12 of the amino acid residues implicated in the catalytic active site. The isolated B. bacilliformis PPase exhibits an activity of 51 ± 2 μmol PO4released/(mg protein∙min) at 28 °C and pH 8, and is sensitive to inhibition by Ca2+. In keeping with other prokaryotic PPases, B. bacilliformis PPase activity occurs from pH 6 to 10 (optimal pH = 8) and demonstrates high thermostability in the presence of Mg2+(highest activity at 55 °C, relative activity = 80 ± 3% at pH 8). The cloned B. bacilliformis ppa is able to genetically complement a ppa−mutant strain of E. coli.Key words: Bartonella, invasion-associated locus, inorganic pyrophosphatase.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals The functional dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels

2004 ◽  
Vol 377 (1) ◽  
pp. 25-36 ◽  
Author(s):  
Stéphanie MOUHAT ◽  
Amor MOSBAH ◽  
Violeta VISAN ◽  
Heike WULFF ◽  
Muriel DELEPIERRE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Scorpion Toxin ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Residues ◽  
Voltage Gated ◽  
Toxin Binding ◽  
Ic50 Values

Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 µg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 µM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

scholarly journals An Evaluation of 3-Rhamnosylquercetin, a Glycosylated Form of Quercetin, against the Myotoxic and Edematogenic Effects of sPLA2fromCrotalus durissus terrificus

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Daniela de Oliveira Toyama ◽  
Henrique Hessel Gaeta ◽  
Marcus Vinícius Terashima de Pinho ◽  
Marcelo José Pena Ferreira ◽  
Paulete Romoff ◽  
...  
Keyword(s):  
Amino Acid ◽  
Docking Studies ◽  
Pharmacological Effects ◽  
Amino Acid Residues ◽  
Substrate Molecule ◽  
Pharmacological Activities ◽  
Potent Inhibition ◽  
Crotalus Durissus

This paper shows the results of quercitrin effects on the structure and biological activity of secretory phospholipase (sPLA2) fromCrotalus durissus terrificus, which is the main toxin involved in the pharmacological effects of this snake venom. According to our mass spectrometry and circular dichroism results, quercetin was able to promote a chemical modification of some amino acid residues and modify the secondary structure ofC. d. terrificussPLA2. Moreover, molecular docking studies showed that quercitrin can establish chemical interactions with some of the crucial amino acid residues involved in the enzymatic activity of the sPLA2, indicating that this flavonoid could also physically impair substrate molecule access to the catalytic site of the toxin. Additionally,in vitroandin vivoassays showed that the quercitrin strongly diminished the catalytic activity of the protein, altered its Vmax and Km values, and presented a more potent inhibition of essential pharmacological activities in theC. d. terrificussPLA2, such as its myotoxicity and edematogenic effect, in comparison to quercetin. Thus, we concluded that the rhamnose group found in quercitrin is most likely essential to the antivenom activities of this flavonoid againstC. d. terrificussPLA2.

Pyruvate kinase from the earthworm Allolobophora calliginosa: modification of the enzyme during anaerobiosis

1991 ◽  
Vol 69 (1) ◽  
pp. 251-254
Author(s):  
Athanasios I. Papadopoulos ◽  
Basile Michaelidis ◽  
Isidoros Beis
Keyword(s):  
Pyruvate Kinase ◽  
Deae Cellulose ◽  
Relative Activity ◽  
The Body ◽  
E Coli ◽  
In Vitro Incubation ◽  
Sigmoidal Kinetics ◽  
Independent Protein

The relative activity of pyruvate kinase from the body-wall muscle of the earthworm Allolobophora calliginosa was found to drop dramatically within 6 h of exposure to N2, whereas the opposite was observed during recovery. Two forms of pyruvate kinase (designated as peak I and peak II) were separated chromatographically on DEAE-cellulose and eluted at 50 and 150 mM of KCl, respectively. They displayed different kinetic behaviour with respect to substrate phosphoenolpyruvate; peak I exhibited Michaelis–Menten kinetics whereas peak II showed sigmoidal kinetics. The ratio of the enzyme units (peak I/peak II) decreased from 3.38 under normoxic conditions to 0.09 under anoxic conditions. In vitro incubation of the aerobic form of pyruvate kinase in the presence of ATP and Mg++ resulted in a reduction of the enzyme activity by 64%, suggesting the presence of an endogenous cyclic-nucleotide-independent protein kinase capable of phosphorylating pyruvate kinase. After in vitro incubation, alkaline phosphatase from E. coli increased the depressed activity of anaerobic pyruvate kinase, indicating that the enzyme molecule is phosphorylated in vivo during exposure to anoxia.

scholarly journals Reconstruction of the immunogenic peptide RNase(43-56) by identification and transfer of the critical residues into an unrelated peptide backbone.

1989 ◽  
Vol 170 (1) ◽  
pp. 203-215 ◽  
Author(s):  
R G Lorenz ◽  
A N Tyler ◽  
P M Allen
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Peptide Backbone ◽  
Chimeric Peptide ◽  
Critical Residues

The involvement of each of the amino acid residues of the I-Ak-restricted T cell determinant RNase(43-56) was examined in detail using a series of peptides containing single amino acid substitutions. Four positions were identified as being essential for the formation of the determinant, Phe-46, Val-47, His-48, and Leu-51. When these four residues were substituted into the backbone of the unrelated peptide HA(130-144), a nonstimulatory peptide was obtained. The inclusion of an additional residue, Val-54, resulted in a chimeric peptide, RN/HA2, which was nearly as active as the native molecule. The peptide RN/HA2 was able to prime in vivo for RNase reactivity, confirming that these five residues contained all of the specificity of the RNase(43-56) determinant. The role of three of these critical residues was examined using both a functional competition assay and an in vivo priming assay. It was ascertained that the Phe-46 was directly involved in contacting the TCR, while the His-48 and Leu-51 were either involved in binding to the I-Ak molecule or in determining the conformation of the peptide. Thus, by critically evaluating the contribution of each of the amino acid residues in a T cell determinant, we were able to generate a chimeric peptide only containing 5 of 15 residues from the RNase(43-56) sequence that was functionally identical to the native RNase(43-56) molecule both in vitro and in vivo.

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