Differences in Transcription and Translation of Long and Short GSα, the Stimulatory G-Protein, in Human Atrium

1995 ◽  
Vol 89 (5) ◽  
pp. 487-495 ◽  
Author(s):  
M. Susan Monteith ◽  
Tao Wang ◽  
Morris J. Brown
Keyword(s):  
Cyclic Amp ◽  
Isoelectric Point ◽  
Right Atrial ◽  
Two Dimensional ◽  
Human Atrium ◽  
Post Translational Modification ◽  
Two Dimensional Gel Electrophoresis ◽  
Significant Difference ◽  
Isoelectric Points

1. We have previously reported the relative mRNA and protein level of the long and short splice variants of Gsα (GsαL and GsαS) in human atrium. We have now measured the relative proportions of the serine+ and serine− variants of GsαL and GsαS in human atrium, and assessed, indirectly, whether their differential expression may (i) regulate Gsα phosphorylation, and (ii) be regulated by atrial cyclic AMP levels. 2. The serine+ and serine− variants of GsαL and GsαS were estimated by single nucleotide primer extension in 36 right atrial strips of which half were from β-adrenoceptor-blocked patients. The ratio of serine+ to serine− variants was 0.06 ± 0.12 for GsαL, compared with 8.04 ± 12.16 for GsαS (P < 0.001). 3. Isoelectric points of GsαL and GsαS in the atria of four β-adrenoceptor-blocked and four non-β-adrenoceptor-blocked patients were estimated by two-dimensional gel electrophoresis. Two-dimensional gel analysis gave a consistent pattern with several spots for both GsαL and GsαS; however, the isoelectric points of GsαS were more acid (5.18 ± 0.24) than those of GsαL (5.87 ± 0.17, P < 0.001). 4. No significant difference in either the serine variants or isoelectric point value was observed between β-adrenoceptor-blocked and non-β-adrenoceptor-blocked patients. 5. In conclusion, all four Gsα variants were expressed in human atrium, but GsαL is almost entirely of the serine− form. GsαS has a more acidic isoelectric point than GsαL, indicating a possible post-translational modification. The lack of difference in our results between β-adrenoceptor-blocked and non-β-adrenoceptor-blocked patients suggests indirectly that cyclic AMP is an unlikely candidate for regulating splicing or post-translational modification of Gsα in vivo.

scholarly journals Altered Protein Expression of Streptococcus oralis Cultured at Low pH Revealed by Two-Dimensional Gel Electrophoresis

2001 ◽  
Vol 67 (8) ◽  
pp. 3396-3405 ◽  
Author(s):  
Joanna C. Wilkins ◽  
Karen A. Homer ◽  
David Beighton
Keyword(s):  
Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Low Ph ◽  
Ionization Mass ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Altered Expression ◽  
Streptococcus Oralis ◽  
Peptide Mass

ABSTRACT Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database forStreptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.

scholarly journals Variations in the Methionine-rich Protein Composition of the Genus Arachis1

1984 ◽  
Vol 11 (1) ◽  
pp. 1-3 ◽  
Author(s):  
Sheikh M. Basha ◽  
Sunil K. Pancholy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Protein Composition ◽  
Electrophoretic Analysis ◽  
Two Dimensional ◽  
Weight Group ◽  
Two Dimensional Gel Electrophoresis ◽  
One Dimensional ◽  
Molecular Weights ◽  
Isoelectric Points

Abstract Methionine-rich proteins (MRP) from seeds of different species of the Genus Arachis were isolated and analyzed by gel electrophoresis to detect possible compositional differences. One-dimensional gel electrophoretic analysis showed presence of quantitative and qualitative variations among the MRP-fractions. Following two-dimensional gel electrophoresis, the MRP-fractions were found to contain three groups of polypeptides with apparent molecular weights of approximately 21,000; 19,000 and 16,000, and isoelectric points between 5.1 and 5.8. Within each molecular weight group the number of polypeptides varied between 1 and 3.

scholarly journals A new variant of the alpha subunit of spectrin in hereditary elliptocytosis

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 473-478 ◽  
Author(s):  
S Lambert ◽  
S Zail
Keyword(s):  
Gel Electrophoresis ◽  
Alpha Subunit ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Tryptic Peptides ◽  
Structural Variant ◽  
Hereditary Elliptocytosis ◽  
Coomassie Blue ◽  
New Variant ◽  
Isoelectric Points

Abstract A kindred is described in which two brothers with a poikilocytic variant of hereditary elliptocytosis (HE) were found to have a defect of spectrin dimer association and a decreased spectrin-band 3 ratio. Two-dimensional gel electrophoresis of limited tryptic digests of their spectrin revealed decreased amounts of the alpha I domain when compared with control digests and the appearance of two major peptides with mol wts of 43,000 and 42,000 and isoelectric points (5.75 to 5.85) more basic than the alpha I domain. Tryptic digests of spectrin from the asymptomatic mother of the two brothers were normal. Immunoblots of the two-dimensional gels using an antiserum to the alpha I domain revealed that the 43,000- and 42,000-dalton peptides were derived from the alpha I domain, along with a series of lower mol wt peptides, some of which were below the detection limits of Coomassie blue-stained gels. Limit chymotryptic maps of 125I-labeled tryptic peptides confirmed that the 43,000- and 42,000-dalton peptides were derived from the alpha I domain. This kindred represents a new structural variant of spectrin in HE in that the major abnormal tryptic peptides derived from the alpha I domain have lower mol wts and more basic isoelectric points than hitherto described.

In vivo distribution of proteins within a single cell.

1982 ◽  
Vol 28 (4) ◽  
pp. 1011-1014 ◽  
Author(s):  
C F Austerberry ◽  
P L Paine
Keyword(s):  
Xenopus Laevis ◽  
Single Cell ◽  
Gel Electrophoresis ◽  
Living Cell ◽  
Experimental System ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
In Vivo Distribution ◽  
Nucleocytoplasmic Distribution

Abstract Using the oocyte of Xenopus laevis, we present an experimental system, involving two-dimensional gel electrophoresis, for measuring unambiguously the nucleocytoplasmic distribution of proteins within a living cell.

scholarly journals Initial Proteome Analysis of Model Microorganism Haemophilus influenzae Strain Rd KW20

2003 ◽  
Vol 185 (15) ◽  
pp. 4593-4602 ◽  
Author(s):  
Eugene Kolker ◽  
Samuel Purvine ◽  
Michael Y. Galperin ◽  
Serg Stolyar ◽  
David R. Goodlett ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Protein Identification ◽  
Proteome Analysis ◽  
Tandem Mass ◽  
Two Dimensional ◽  
High Confidence ◽  
Two Dimensional Gel Electrophoresis

ABSTRACT The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism of H. influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) of H. influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.

scholarly journals Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Effect of adenosine 3′:5′-cyclic monophosphate

1979 ◽  
Vol 184 (2) ◽  
pp. 233-244 ◽  
Author(s):  
S Roberts ◽  
B S Morelos
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Ribosomal Proteins ◽  
Phosphodiesterase Inhibitor ◽  
Distinct Species ◽  
Rat Cerebral Cortex ◽  
Neural Activation ◽  
Two Dimensional ◽  
Basic Proteins

Investigations were carried out on the phosphorylation of ribosomal proteins in vivo in cerebral cortices of immature rats. Two-dimensional electrophoresis revealed that the cerebral 40S subunit contained at least four ribosomal proteins which were phosphorylated in animals given [32P]orthophosphate intracisternally. These proteins exhibited electrophoretic properties similar to those of the constitutive basic proteins S2, S3a, S5 and S6. The cerebral 60S subunit contained several proteins that were phosphorylated in vivo, including three basic proteins with electrophoretic mobilities similar to those of ribosomal proteins L6, L14 and L19. Four other proteins associated with the 60S subunit that were more acidic were also phosphorylated. Phosphorylated congeners of 40S and 60S ribosomal proteins could often be detected in distinct protein-stained spots on two-dimensional electrophoretograms. The cerebral S6 protein consisted of at least five distinct species in different states of phosphorylation. Administration of N6O-2′ dibutyryl cyclic AMP increased the proportion of the more phosphorylated congeners of the S6 protein, but appeared to have little or no effect on phosphorylation of other cerebral ribosomal proteins. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated S6-protein phosphorylation; N2O2′-dibutyryl cyclic GMP had no effect on this process. These observations indicate that several ribosomal proteins of both subunits are normally phosphorylated in rat cerebral cortex in situ. The results also suggest that selective and specific alterations in the phosphorylation state of the S6 ribosomal protein of the cerebral 40S subunit may accompany the production of cyclic AMP during neural activation.

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