Adenosine A2a receptors are found in coronary vascular tissue although, their presence in myocardium is subject to investigation. Although there have been numerous studies on adenosine A2a receptor agonist effects on contractility and cAMP levels in ventricular myocytes, these have yielded conflicting results. Negative pharmacological studies have even led to the conclusion that A2a receptors are not present in cardiac myocytes. The purpose of this study was to determine whether A2a receptors are expressed in rat ventricular myocytes and what physiological effects are mediated via activation of these receptors. Western blot analysis with a polyclonal antibody raised against a peptide sequence specific to the carboxy terminus of the A2a receptor revealed the presence of a band at approximately 45 kDa. However, the immunoreactivity was located in the nonmembrane fraction of the cell lysate. The membrane fraction only exhibited an immunoreactive band > or = 50 kDa. Treatment of isolated myocytes with the adenosine A2a agonist 2-[4-[(2-carboxyethyl)-phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680) exerted no effects on cAMP levels or myocyte twitch amplitude. These results indicate that although rat ventricular myocytes appear to express adenosine A2a receptors, stimulation with an A2a agonist exerts no functional effects, possibly because of the subcellular localization of the A2a receptor.

No abstract

Selenoprotein P (SePP) is a unique selenium-containing protein responsible for the transport and distribution of the essential trace element selenium (Se) through the human body with the concentration of SePP in human blood representing the most useful marker of Se nutritional status. Although SePP has been extensively studied, the structure of SePP in human plasma remains unresolved. Two potential isoforms of SePP have been identified by Western blot analyses distinguished principally by differences in migration (51 kDa and 61 kDa). The biological relevance of the smaller isoform has been called into question by several studies reporting only one major SePP form (69 kDa) suggesting that the shorter 51 kDa is an artifact of protease activity during the SePP purification process. A deficiency of these Western blot analyses is that no information can be gleaned regarding the Se content of the potential isoforms. This study reports a characterization of SePP isoforms in a human plasma Standard Reference Material representative of a healthy US population. Following immunoprecipitation, three SePP isoforms were unequivocally identified at 45 kDa, 49 kDa and 57 kDa (termed as SePP45, SePP49 and SePP57) by LC-MS/MS analyses from a spectral searching approach. Selenium (Se) was detected by gel electrophoresis LA-ICP-MS in SePP49 and SePP57 which was confirmed by the identification of three selenopeptides covering the SePP sequence from residues 312-346 by LC-MS/MS analyses utilizing a sequence searching approach. Conversely, neither Se nor peptides covering SePP sequence from residues 306-346 was identified in SePP45 which suggests that SePP45 is a truncated isoform transcriptionally terminated at the 2nd in-frame UGA codon thereby terminating the protein with the Ser residue at position 299. An additional band at 23 kDa was found to contain Se but no peptides of SePP. Instead, glutathione peroxidase 3 (GPx3) was unequivocally identified within the band presumably being co-immunoprecipitated with the SePP providing preliminary evidence that SePP and GPx3 interaction may take place in vivo.

Between 1 and 2% of proteins coded for in the human genome, including all G protein ␥ subunits, are predicted to be prenylated. Subsequently, prenylated proteins are proteolytically cleaved at the C terminus and carboxymethylated. These reactions are generally obligatory events required for functional expression of prenylated proteins. The biological role of prenyl substrates has made these reactions significant targets for anticancer drug development. Understanding the enzymology of this pathway will be key to success for this strategy. When G␥1, -2, -4, -10, -11, -12, and -13 were expressed in HEK293 cells they were completely processed according to the current understanding of the prenylation reaction. In contrast, G␥5 was processed to two forms; a minor one, fully processed as predicted, and a major one that was prenylated without further processing. When the Ca 1 a 2 X motif of G␥5, CSFL, was exchanged for that of G␥2, CAIL, G␥5 was completely processed. Conversely, G␥2-SFL was incompletely processed. Differential processing of G␥5 was found due to the presence of an aromatic amino acid in its Ca 1 a 2 X motif. Retrieving endogenous G␥ subunits from HEK293 or Neuro-2a cells with FLAG-G␤ constructs identified multiple G␥ subunits by mass spectrometry in either cell, but in both cases the most prominent one was G␥5 expressed without C-terminal processing after prenylation. This work indicates that post-prenylation reactions can generate multiple products determined by the C-terminal Ca 1 a 2 X motif. Within the human genome 10% of predicted prenylated proteins have aromatic amino acids in their Ca 1 a 2 X sequence and would likely generate the prenylation pattern described here.

This paper describes the development of a reference measurement procedure to quantify human C-reactive protein (CRP) in serum using affinity techniques prior to tryptic digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the certification of reference materials in clinically relevant ranges. The absence of a suitable internal standard for the CRP measurement, necessary to eliminate potential measurement bias in both the affinity purification and trypsin digestion steps, was addressed using the method of standard addition. The standard addition quantification approach was combined with affinity purification, using an anti-CRP monoclonal antibody conjugated to polystyrene beads, trypsin digestion of the purified protein, and LC-MS/MS analysis of CRP tryptic peptides. The effectiveness of intact protein affinity purification was evaluated through the measurement of CRP in several serum-based CRP control materials, yielding levels that were comparable to their expected mean concentration values. Quantitative results were confirmed with an external calibration approach. This study demonstrates the feasibility of affinity purification with LC-MS/MS for the reference measurement procedure development of low abundance serum protein analytes.Reference measurement procedures are used for a variety of purposes including (1) the assessment of the measurement quality of routine measurement procedures, (2) the value assignment of high-order calibration solutions (the so-called "master calibrators") for routine assays, and (3) the value assignment of certified reference materials. 1,2 In these roles, reference measurement procedures are considered "higher-order" measurement procedures, as they aim to achieve a higher level of accuracy and precision than the routine assay for the analyte being measured, producing a value with a low uncertainty of defined magnitude. In order for a reference measurement procedure to be fit for this purpose, it must be thoroughly evaluated to identify sources of bias and assess their magnitude. [3][4][5] Minimizing or eliminating bias is necessary to measure the most accurate, true concentration of the analyte. Because of the need for high levels of accuracy and precision, reference measurement procedures are typically low-throughput, labor intensive, and often costly to perform, in sharp contrast to what is desirable for routine measurements.Reference measurement procedures are key elements in the establishment of measurement standardization and metrological traceability in clinical chemistry. For more than 30 years, isotope dilution mass spectrometry (IDMS) has been one of the analytical techniques used frequently in reference measurement procedures, particularly for small molecule and inorganic analytes of clinical importance. 6 Despite this, few reference measurement procedures exist for clinically relevant proteins. This is one reason why so many clinical protein analytes are measured in arbitrary International Units (IUs); without a reference measurement procedure to...