scholarly journals HPLC Method to Resolve, Identify and Quantify Guanine Nucleotides Bound to the GTPase Ras

2021 ◽  
Author(s):  
Jonathan P Hannan ◽  
George Hayden Swisher ◽  
Justin G Martyr ◽  
Nicholas J Cordaro ◽  
Annette H Erbse ◽  
...  
Keyword(s):  
G Protein ◽  
Nucleotide Composition ◽  
Hplc Method ◽  
Heat Extraction ◽  
Disease Mechanism ◽  
Reverse Phase Hplc ◽  
Functional Studies ◽  
Back Calculation ◽  
The Individual

The Ras superfamily of small G proteins play central roles in diverse signaling pathways. Superfamily members act as molecular on-off switches defined by their occupancy with GTP or GDP, respectively. In vitro functional studies require loading with a hydrolysis-resistant GTP analogue to increase the on-state lifetime, as well as knowledge of fractional loading with activating and inactivating nucleotides. The present study describes a method combining elements of previous approaches with new, optimized features to analyze the bound nucleotide composition of a G protein loaded with activating (GMPPNP) or inactivating (GDP) nucleotide. After nucleotide loading, the complex is washed to remove unbound nucleotides then bound nucleotides are heat-extracted and subjected to ion-paired, reverse-phase HPLC-UV to resolve, identify and quantify the individual nucleotide components. These data enable back-calculation to the nucleotide composition and fractional activation of the original, washed G protein population prior to heat extraction. The method is highly reproducible. Application to multiple HRas preparations and mutants confirms its ability to fully extract and analyze bound nucleotides, and to resolve the fractional on- and off-state populations. Furthermore, the findings yield a novel hypothesis for the molecular disease mechanism of Ras mutations at the E63 and Y64 positions.

scholarly journals POLRMT mutations impair mitochondrial transcription causing neurological disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Monika Oláhová ◽  
Bradley Peter ◽  
Zsolt Szilagyi ◽  
Hector Diaz-Maldonado ◽  
Meenakshi Singh ◽  
...  
Keyword(s):  
Progressive External Ophthalmoplegia ◽  
Global Developmental Delay ◽  
Disease Mechanism ◽  
Mitochondrial Transcription ◽  
Functional Studies ◽  
Mtdna Deletions ◽  
Molecular Nature ◽  
Important Disease

AbstractWhile >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

Validation and application of a simple reverse phase HPLC method for in vitro dissolution studies of memantine hydrochloride tablet

2015 ◽  
Vol 45 (5) ◽  
pp. 415-421 ◽  
Author(s):  
Han-Joo Maeng ◽  
Sung-Up Choi ◽  
Dong-Jin Jang ◽  
Dong Won Lee ◽  
Byung-Nak Ahn ◽  
...  
Keyword(s):  
Hplc Method ◽  
In Vitro Dissolution ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Dissolution Studies

scholarly journals GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 5984
Author(s):  
Philip Rößler ◽  
Daniel Mayer ◽  
Ching-Ju Tsai ◽  
Dmitry B. Veprintsev ◽  
Gebhard F. X. Schertler ◽  
...  
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Conformational Ensemble ◽  
Gpcr Activation ◽  
Relevant Situation ◽  
Gs Protein ◽  
The Individual ◽  
G Protein Coupled ◽  
Activation Cycle

In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[13CH3] labelled thermostabilized β1 adrenergic receptor from turkey in association with a variety of different effectors, including mini-Gs and nanobody 60 (Nb60), which have not been previously studied in complex with β1 adrenergic receptor (β1AR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of tβ1AR stabilized by the native-like mini-Gs protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.

Structural and Functional Alterations in the Mucosa of Self-Filling Intestinal Blind Loops in Rats

1979 ◽  
Vol 56 (2) ◽  
pp. 121-131 ◽  
Author(s):  
H. Menge ◽  
R. Köhn ◽  
K.-H. Dietermann ◽  
H. Lorenz-Meyer ◽  
E. O. Riecken ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Blind Loop ◽  
Rat Colon ◽  
Jejunal Mucosa ◽  
Functional Studies ◽  
Intestinal Contents ◽  
The Individual ◽  
Lower Ileum ◽  
Esterase Activities

1. Self-filling blind loops of jejunum were constructed in three groups of rats; in the first, blind loops were created without further manipulation; in the second the bile was diverted permanently into the lower ileum below the blind loop, whereas in a third neomycin was added to the drinking water throughout the experiment. Two weeks after the creation of the blind loops, they were used for structural and functional studies. 2. Morphometric and microdissection techniques demonstrated that the surface area of the individual villi of the mucosa of ‘ordinary’ blind loops had increased fourfold in comparison with corresponding control jejunum, whereas the increase was only twofold in rats with bile diversion or in the series treated with neomycin. There were proportional increases in crypt length and mitotic activity of the crypts in all three series, which suggest that the alterations in the mucosa were due to hyperplasia in both villus and crypt compartments. 3. Sucrase, succinate dehydrogenase, alkaline phosphatase and non-specific esterase activities, determined biochemically or histochemically, were reduced in the mucosae of all blind loops, though the changes were most pronounced in the ‘ordinary’ blind loops. The accumulation of l-phenylalanine by mucosal slices in vitro was depressed, although the decrease was less marked in the series treated with neomycin. 4. These results suggest that both bacteria and deconjugated bile acids play a role in the development of the hyperplastic changes of the blind-loop mucosa, but that another factor might also be involved: as a possible candidate, stasis of the intestinal contents was considered. 5. To test this hypothesis, loops of rat colon were transposed into the jejunum. Above the transposed loop, the jejunal mucosa developed hyperplasia of both villus and crypt compartments, with a reduction in its ability to accumulate l-phenylalanine. It is argued that these changes, probably caused by stasis of the intestinal contents, are triggered off by the dilatation of the gut, which may also be implicated in the mucosal alterations in blind loops.

scholarly journals Reverse-phase HPLC of the hydrophobic pulmonary surfactant proteins: detection of a surfactant protein C isoform containing Nε-palmitoyl-lysine

1997 ◽  
Vol 326 (3) ◽  
pp. 799-806 ◽  
Author(s):  
Magnus GUSTAFSSON ◽  
Tore CURSTEDT ◽  
Hans JÖRNVALL ◽  
Jan JOHANSSON
Keyword(s):  
Pulmonary Surfactant ◽  
Surfactant Protein ◽  
Fatty Acyl ◽  
Hplc Method ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Functional Studies ◽  
Hydrophobic Peptides ◽  
Major Form ◽  
Pulmonary Surfactant Proteins

A reverse-phase HPLC protocol for analysis of strictly hydrophobic peptides and proteins was developed. Peptide aggregation is minimized by using only 25–40% water in methanol or ethanol as initial solvents and subsequent elution with a gradient of propan-2-ol. Analysis of the pulmonary surfactant-associated proteins B (SP-B) and C (SP-C) with this method reveals several features. (1) SP-B and SP-C retain their secondary structures and separate by about 15 min over a 40 min gradient. SP-B is more hydrophilic than SP-C, which in turn behaves chromatographically like palmitoyl-ethyl ester. (2) SP-C exhibits isoforms additional to the major form characterized previously, which contains two thioester-linked palmitoyl groups. The isoforms now observed contain one or three palmitoyl moieties and constitute together 15–20% of the major form. The tripalmitoylated species contains a palmitoyl group linked to the ϵ-amino group of Lys-11, as concluded from the elution position, MS and amino acid sequence analysis. The tripalmitoylated form increases relative to the dipalmitoylated form on incubation of SP-C in a phospholipid environment. An Nϵ-bound palmitoyl moiety constitutes a third mode of fatty acyl modification of proteins, in addition to the established Nα-bound myristoyl groups and S-bound palmitoyl chains. (3) The dimeric structure of SP-B, lacking covalent modifications, is confirmed by MS detection of the dimer. No SP-B isoforms were detected. (4) Denatured, non-helical SP-C can be distinguished chromatographically from the native α-helical peptide. (5) HPLC of SP-C at 60–75 °C reveals an isoform containing an extra 14 Da moiety compared with the main form. This is concluded to arise from inadvertent methyl esterification of the C-terminal carboxy group. In conclusion, this HPLC method affords a sensitive means of assessing modifications and conformations of SP-B or SP-C in different disease states and before functional studies. It might also prove useful for analysis of other strictly hydrophobic polypeptides.

scholarly journals Molecular analysis of an enigmatic Streptococcus pneumoniae virulence factor: The raffinose-family oligosaccharide utilization system

2019 ◽  
Vol 294 (46) ◽  
pp. 17197-17208 ◽  
Author(s):  
Joanne K. Hobbs ◽  
Edward P. W. Meier ◽  
Benjamin Pluvinage ◽  
Mackenzie A. Mey ◽  
Alisdair B. Boraston
Keyword(s):  
Streptococcus Pneumoniae ◽  
Catalytic Efficiency ◽  
Respiratory Pathogen ◽  
Tissue Tropism ◽  
Raffinose Family Oligosaccharides ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Wide Range ◽  
The Individual

Streptococcus pneumoniae is an opportunistic respiratory pathogen that can spread to other body sites, including the ears, brain, and blood. The ability of this bacterium to break down, import, and metabolize a wide range of glycans is key to its virulence. Intriguingly, S. pneumoniae can utilize several plant oligosaccharides for growth in vitro, including raffinose-family oligosaccharides (RFOs, which are α-(1→6)-galactosyl extensions of sucrose). An RFO utilization locus has been identified in the pneumococcal genome; however, none of the proteins encoded by this locus have been biochemically characterized. The enigmatic ability of S. pneumoniae to utilize RFOs has recently received attention because mutations in two of the RFO locus genes have been linked to the tissue tropism of clinical pneumococcal isolates. Here, we use functional studies combined with X-ray crystallography to show that although the pneumococcal RFO locus encodes for all the machinery required for uptake and degradation of RFOs, the individual pathway components are biochemically inefficient. We also demonstrate that the initiating enzyme in this pathway, the α-galactosidase Aga (a family 36 glycoside hydrolase), can cleave α-(1→3)-linked galactose units from a linear blood group antigen. We propose that the pneumococcal RFO pathway is an evolutionary relic that is not utilized in this streptococcal species and, as such, is under no selection pressure to maintain binding affinity and/or catalytic efficiency. We speculate that the apparent contribution of RFO utilization to pneumococcal tissue tropism may, in fact, be due to the essential role the ATPase RafK plays in the transport of other carbohydrates.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

Identification of new ARMC5 missense mutations in Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) and their functional studies in vitro

2018 ◽  
Author(s):  
Anna Vaczlavik ◽  
Stephanie Espiard ◽  
Marie-Odile North ◽  
Ludivine Drougat ◽  
Marthe Rizk-Rabin ◽  
...  
Keyword(s):  
Missense Mutations ◽  
Adrenal Hyperplasia ◽  
Functional Studies

In-vitro Kinetic Hydrolysis Study of Metronidazole Derivatives with Carvacrol and Eugenol Using Validated RP-HPLC Method

2020 ◽  
Vol 16 ◽  
Author(s):  
M. Alarjah
Keyword(s):  
Half Life ◽  
Hplc Method ◽  
Hydrolysis Rate ◽  
First Order ◽  
First Order Kinetics ◽  
Rp Hplc ◽  
Pharmacokinetic Properties ◽  
Pseudo First Order ◽  
Kinetic Hydrolysis

Background: Prodrugs principle is widely used to improve the pharmacological and pharmacokinetic properties of some active drugs. Much effort was made to develop metronidazole prodrugs to enhance antibacterial activity and or to improve pharmacokinetic properties of the molecule or to lower the adverse effects of metronidazole. Objective: In this work, the pharmacokinetic properties of some of monoterpenes and eugenol pro metronidazole molecules that were developed earlier were evaluated in-vitro. The kinetic hydrolysis rate constants and half-life time estimation of the new metronidazole derivatives were calculated using the validated RP-HPLC method. Method: Chromatographic analysis was done using Zorbbax Eclipse eXtra Dense Bonding (XDB)-C18 column of dimensions (250 mm, 4.6 mm, 5 μm), at ambient column temperature. The mobile phase was a mixture of sodium dihydrogen phosphate buffer of pH 4.5 and methanol in gradient elution, at 1ml/min flow rate. The method was fully validated according to the International Council for Harmonization (ICH) guidelines. The hydrolysis process carried out in an acidic buffer pH 1.2 and in an alkaline buffer pH 7.4 in a thermostatic bath at 37ºC. Results: The results followed pseudo-first-order kinetics. All metronidazole prodrugs were stable in the acidic pH, while they were hydrolysed in the alkaline buffer within a few hours (6-8 hr). The rate constant and half-life values were calculated, and their values were found to be 0.082- 0.117 hr-1 and 5.9- 8.5 hr., respectively. Conclusion: The developed method was accurate, sensitive, and selective for the prodrugs. For most of the prodrugs, the hydrolysis followed pseudo-first-order kinetics; the method might be utilised to conduct an in-vivo study for the metronidazole derivatives with monoterpenes and eugenol.

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