A 4,000-dalton dynorphin was isolated from porcine pituitary. It has 32 amino acids (Mr= 3,986), with the previously described heptadecapeptide (now called dynorphin A) at its amino terminus and a ...related tridecapeptide, dynorphin B, at its carboxyl terminus. The two peptides are separated by the ``processing signal'' Lys-Arg.
The gene encoding glucose transporter 3 (GLUT3, SLC2A3) is present in the human population at variable copy number. An overt disease phenotype of SLC2A3 copy number variants has not been reported; ...however, deletion of SLC2A3 has been previously reported to protect carriers from rheumatoid arthritis, implicating GLUT3 as a therapeutic target in rheumatoid arthritis. Here we aim to perform functional analysis of GLUT3 copy number variants in immune cells, and test the reported protective association of the GLUT3 copy number variants for rheumatoid arthritis in a genetic replication study.
Cells from genotyped healthy controls were analyzed for SLC2A3/GLUT3 expression and glycolysis capacity. We genotyped the SLC2A3 copy number variant in four independent cohorts of rheumatoid arthritis and controls and one cohort of multiple sclerosis and controls.
Heterozygous deletion of SLC2A3 correlates directly with expression levels of GLUT3 and influences glycolysis rates in the human immune system. The frequency of the SLC2A3 copy number variant is not different between rheumatoid arthritis, multiple sclerosis and control groups.
Despite a robust SLC2A3 gene copy number dependent phenotype, our study of large groups of rheumatoid arthritis cases and controls provides no evidence for rheumatoid arthritis disease protection in deletion carriers. These data emphasize the importance of well powered replication studies to confirm or refute genetic associations, particularly for relatively rare variants.
•T cell and macrophage expression of SLC2A3/GLUT3 correlates to SLC2A3 gene copy number in a dose dependent manner.•Glycolysis rates are reduced in individuals harboring a deletion of the GLUT3 gene SLC2A3•Deletion of SLC2A3 is not associated with protection from rheumatoid arthritis•Deletion of SLC2A3 is not associated with risk for multiple sclerosis•GLUT3 is not a viable therapeutic target for RA as previously proposed based on a protective association of SLC2A3 deletion.
The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli undergo reversible methylation that has been correlated with adaptation of cells to environmental stimuli. MCPI, the product of the ...tsr gene, accepts methyl groups at multiple sites that are located on two tryptic peptides, denoted K1 and R1. A second modification of the MCPs, which is not methylation, has been designated the CheB-dependent modification. A CheB-dependent modification occurs on methyl-accepting peptide K1 and allows additional methyl groups to be incorporated into this peptide. We have performed partial amino acid sequence analyses on radiolabeled peptides K1 and R1 derived from MCPI and have identified several methyl-accepting sites. We found that, in the absence of CheB-dependent modification, a site in peptide K1 is unable to accept methyl groups. Correlation of this protein sequence data with the nucleotide sequence of the tsr gene Boyd, A., Kendall, K. & Simon, M. I. (1983) Nature (London) 301, 623-626 suggests that CheB-dependent modification of MCPI is the enzymatic deamidation of glutamine to methyl-accepting glutamic acid. Possible roles for this deamidation in bacterial chemotaxis are discussed.
Contemporary Methodology for Protein Structure Determination Hunkapiller, Michael W.; Strickler, James E.; Wilson, Kenneth J.
Science (American Association for the Advancement of Science),
1984-Oct-19, Volume:
226, Issue:
4672
Journal Article
Peer reviewed
The techniques used for the characterization of protein and peptide structure have undergone great changes that have improved the speed, reliability, and applicability of the process. ...High-performance liquid chromatography and gel electrophoresis have made the purification of proteins and peptides a routine procedure, even when the compound of interest is a minor component of a complex biological mixture. The chemistry and instrumentation used in amino acid analysis and amino acid sequencing now permit the analysis of as little as 5 to 50 picomoles of samples. This represents an increase in sensitivity of more than a thousandfold over the last 10 years and has made possible the structural analysis of a wide variety of scarce but important compounds.
Stopped-flow spectrophotometry and proton inventory experiments have been used to define the reaction pathway for hydrolysis of a specific peptide substrate, Ac-L-Ala-L-Pro-L-Ala p-nitroanilide, by ...the serine proteases elastase and alpha-lytic protease. The stopped-flow studies reveal the existence and buildup of a tetrahedral adduct between the active site serine hydroxyl group and the sensitive carbonyl group of the substrate. The decomposition of this tetrahedral intermediate to the acyl enzyme and p-nitroaniline is the rate-limiting step for the hydrolytic reaction. The proton inventory data suggest the simultaneous transfer of two protons (presumably from the catalytic carboxyl of Asp-102 to N pi of the catalytic imidazole of His-57 and from N pi of the imidazole to the anilide NH) in the transition state leading to breakdown of the tetrahedral complex. That these proton transfers occur in a concerted, rather than stepwise, process attests to the ability of enzymes to lower the enthalpy of activation most effectively when the precise alignment of a highly specific substrate and catalytic groups minimizes the entropy of activation.
Homogeneous human lymphoblastoid interferon with an apparent molecular size of 18,500 daltons was characterized by its amino acid composition. Analysis of the amino terminal sequence by Edman ...degradation indicates that the sequence is unique.
A procedure utilizing reversed-phase high-performance liquid chromatography is described for the purification of asialo granulocyte--macrophage colony stimulating factor (asialo-GM-CSF) from mouse ...lung-conditioned medium. In the purification, the partially purified factor was treated with neuraminidase to reduce charge heterogeneity due to variable degrees of sialation. Three active forms of the asialo factor were separated by the final reversed-phase liquid chromatography step. These each gave a single major band and several minor bands on polyacrylamide gel electrophoresis and had similar amino acid compositions. The specific activity of purified murine asialo-GM-CSF was approximately 8 × 109colonies per mg of protein. Amino acid sequence determination of the major form gave a single amino-terminal sequence, which has been used to develop oligonucleotide probes for the isolation of two cDNA clones encoding GM-CSF. The nucleotide sequence of these two clones gave a deduced amino acid sequence almost identical with that determined for the amino terminus of asialo-GM-CSF and an amino acid composition very similar to that for asialo-GM-CSF.
The nicotinic acetylcholine receptor has been purified from fetal calf muscle. Amino terminal amino acid sequence data indicate that the mammalian receptor is formed from closely related but distinct ...subunits. A cytoskeletal component, actin, may be associated with the receptor.
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