PubMed · 4504108
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J Folch-Pi, P J Stoffyn. 1972-06-20. Proteolipids from membrane systems.. https://pubmed.ncbi.nlm.nih.gov/4504108/
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Amino acid (AA) profiles from body fluids such as blood and urine are clinical indicators for diagnosing metabolic and hepatic diseases. Current quantitative methods, such as liquid chromatography-mass spectrometry (LC-MS) with isotopically labelled internal standards (ISs), are costly and technically demanding. This study proposes a cost-efficient alternative using structural isomers as ISs in a direct liquid infusion (DLI) tandem mass spectrometry (MS/MS) approach. The method leverages chimeric spectra and fragment intensity ratios to quantify AAs, demonstrating high linearity and precision even with a 3D ion trap mass analyser. This approach offers a viable strategy for AA quantification in preventive medicine, particularly for screening metabolic diseases such as phenylketonuria, diabetes, and liver dysfunction.
Herein, we report on the mutational analysis of a 70-amino acid segment (region 1, residues 438-508) of family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol). Region 1, which lies between the Exo III sequence and the similarity motif D- -SLYP, connects the exonuclease and polymerase domains of Sso DNA pol. Two C-terminally deleted forms of the enzyme, proteins N438 (residues 1-438) and N508 (residues 1-508), were overproduced in the recombinant form and biochemically characterized. They contain the three evolutionarily conserved Exo motifs, but differ in the extent of the C-terminal deletion, since only N508 includes region 1. Both have been found to retain a Mn2+-dependent 3'-5' exonuclease activity, whose thermal stability appears to be increased in comparison to that of the full-sized enzyme. Assays for processive 3'-5' exonuclease activity, carried out with the heparin trap method on a 24-base oligonucleotide, have revealed that protein N508, as well as the full-length Sso DNA pol, retains a level of processivity of the degradative function substantially higher than that for protein N438. In addition, six site-specific mutations have been introduced at the highly conserved Y-GG/A motif, which has been found within Sso DNA pol region 1. All mutant proteins (Lys491Ile, Tyr495Ser, Lys496Ile, Gly497Ala, and Ala498Val) display increased processivity of their 3'-5' exonuclease activity, with the exception of protein Tyr495Phe. By a steady-state kinetic analysis of the exonucleolytic reaction on a 24-base oligonucleotide, the above site-specific mutations have been found to affect Km values consistently with the observed differences in the processivity values, whereas the effect on the kcat values seems to be less important. The results from this mutational analysis indicate that region 1 is involved in determining the processivity of the proofreading function, directly interacting with the nucleic acid substrate.
We report the isolation and the functional characterization of alpha and beta chains from pig (Sus scropha domesticus) haemoglobin, as well as of the pig-human hybrid haemoglobins, alpha2(h)beta2(p) and alpha2(p)beta2(h) (i.e. Circe's haemoglobins), obtained by mixing the purified alpha and beta pig chains respectively with the corresponding partner human chains. Their functional properties have been compared with those of both parental haemoglobins in order to obtain information on the role of the different subunits and of their inter-relationships, both at the structural and functional levels. The results indicate that the functional properties of both hybrids are closer to those of the parental haemoglobin that provides the beta chains, confirming the major role of the beta chains in determining the oxygen affinity and the modulation mechanisms of the tetrameric molecule. This is supported by the thermodynamic properties, since the very low DeltaH of oxygen binding that characterizes pig haemoglobin and the alpha2(h)beta2(p) hybrid haemoglobin may be taken as the reflection of specific structural properties of pig beta chain.