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  • Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function. 24361330

    Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.
    Document Type:
    Reference
    Product Catalog Number:
    07-375
    Product Catalog Name:
    Anti-Ubiquitin Antibody

  • Water in Alanine

    Document Type:
    Application
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by a ... 11964291

    In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor in determining their efficacy because evidence from antibody-blocking studies indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs is demonstrated. Mutation of alanine and proline at positions 170 and 172 (AxP) (single-letter amino acid codes; x indicates the identical amino acid at the same position in the murine and human CD20 sequences) in human CD20 abrogated the binding of all CD20 mAbs tested. Introduction of AxP into the equivalent positions in the murine sequence, which is not otherwise recognized by antihuman CD20 mAbs, fully reconstituted the epitope recognized by B1, the prototypic anti-CD20 mAb. 2H7, a mAb previously thought to recognize the same epitope as B1, did not recognize the murine AxP mutant. Reconstitution of the 2H7 epitope was achieved with additional mutations replacing VDxxD in the murine sequence for INxxN (positions 162-166 in the human sequence). The integrity of the 2H7 epitope, unlike that of B1, further depends on the maintenance of CD20 in an oligomeric complex. The majority of 16 antihuman CD20 mAbs tested, including rituximab, bound to murine CD20 containing the AxP mutations. Heterogeneity in the fine specificity of these antibodies was indicated by marked differences in their ability to induce homotypic cellular aggregation and translocation of CD20 to a detergent-insoluble membrane compartment previously identified as lipid rafts.
    Document Type:
    Reference
    Product Catalog Number:
    MABF250

  • STE20/SPS1-related proline/alanine-rich kinase is involved in plasticity of GABA signaling function in a mouse model of acquired epilepsy. 24058604

    The intracellular concentration of chloride ([Cl(-)]i) determines the strength and polarity of GABA neurotransmission. STE20/SPS1-related proline/alanine-rich kinase (SPAK) is known as an indirect regulator of [Cl(-)]i for its activation of Na-K-2 Cl(-)co-transporters (NKCC) and inhibition of K-Cl(-)co-transporters (KCC) in many organs. NKCC1 or KCC2 expression changes have been demonstrated previously in the hippocampal neurons of mice with pilocarpine-induced status epilepticus (PISE). However, it remains unclear whether SPAK modulates [Cl(-)]i via NKCC1 or KCC2 in the brain. Also, there are no data clearly characterizing SPAK expression in cortical or hippocampal neurons or confirming an association between SPAK and epilepsy. In the present study, we examined SPAK expression and co-expression with NKCC1 and KCC2 in the hippocampal neurons of mice with PISE, and we investigated alterations in SPAK expression in the hippocampus of such mice. Significant increases in SPAK mRNA and protein levels were detected during various stages of PISE in the PISE mice in comparison to levels in age-matched sham (control) and blank treatment (control) mice. SPAK and NKCC1 expression increased in vitro, while KCC2 was down-regulated in hippocampal neurons following hypoxic conditioning. However, SPAK overexpression did not influence the expression levels of NKCC1 or KCC2. Using co-immunoprecipitation, we determined that the intensity of interaction between SPAK and NKCC1 and between SPAK and KCC2 increased markedly after oxygen-deprivation, whereas SPAK overexpression strengthened the relationships. The [Cl(-)]i of hippocampal neurons changed in a corresponding manner under the different conditions. Our data suggests that SPAK is involved in the plasticity of GABA signaling function in acquired epilepsy via adjustment of [Cl(-)]i in hippocampal neurons.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide ass ... 2574667

    1. We have synthesized two peptides, one based on the exact sequence around the unique site (Ser79) for the AMP-activated protein kinase on rat acetyl-CoA carboxylase (SSMS peptide) and another in which the serine residue corresponding to the site for cyclic-AMP-dependent protein kinase (Ser77) was replaced by alanine (SAMS peptide). 2. Both peptides were phosphorylated with similar kinetics by the AMP-activated protein kinase, but only the SSMS peptide was a substrate for cyclic-AMP-dependent protein kinase. The SAMS peptide was not phosphorylated by any of five other purified protein kinases tested. 3. The Km of AMP-activated protein kinase for the SAMS peptide is higher than that for acetyl-CoA carboxylase, but the Vmax for peptide phosphorylation is 2.5 times higher than that of its parent protein. This peptide therefore gives a convenient and sensitive assay for the AMP-activated protein kinase. 4. Acetyl-CoA-carboxylase kinase and peptide kinase activities copurify through six steps from a post-mitochondrial supernatant of rat liver, showing that the SAMS peptide is a specific substrate for the AMP-activated protein kinase in this tissue. We could not demonstrate AMP-dependence of the kinase activity in crude preparations, apparently due to endogenous AMP remaining bound to the enzyme. However, 8-bromoadenosine 5-monophosphate (Br8AMP) is a partial agonist at the allosteric (AMP) site, and inhibition by 2 mM Br8AMP can be used to test that one is measuring the AMP-stimulated form of the kinase. 5. Using this approach, we have examined the kinase activity in nine different rat tissues, plus a mouse macrophage cell line, and find that there is a correlation between tissues expressing significant levels of peptide kinase activity and those active in the synthesis or storage of lipids. 6. We also use the peptide assay to show that cyclic AMP-dependent protein kinase does not activate purified AMP-activated protein kinase, and does not affect the activation of partially purified AMP-activated protein kinase by endogenous kinase kinase.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases. 2108169

    Acetylation of Ser-530 of sheep prostaglandin endoperoxide (PGG/H) synthase by aspirin causes irreversible inactivation of the cyclooxygenase activity of the enzyme. To determine the catalytic function of the hydroxyl group of Ser-530, we used site-directed mutagenesis to replace Ser-530 with an alanine. Cos-1 cells transfected with expression vectors containing the native (Ser-530) or mutant (Ala-530) cDNAs for sheep PGG/H synthase expressed comparable cyclooxygenase and hydroperoxidase activities. Km values for arachidonate (8 microM) and ID50 values for reversible inhibition by the cyclooxygenase inhibitors, flurbiprofen (5 microM), flufenamate (20 microM), and aspirin (20 mM), were also the same for both native and mutant PGG/H synthases; however, only the native enzyme was irreversibly inactivated by aspirin. Thus, the "active site" Ser-530 of PGG/H synthase is not essential for catalysis or substrate binding. Apparently, acetylation of native PGG/H synthase by aspirin introduces a bulky sidechain at position 530 which interferes with arachidonate binding. In related studies, a cDNA for mouse PGG/H synthase was cloned and sequenced. A sequence of 35 residues with Ser-530 at the midpoint was identical in the two proteins. Thus, Ser-530 does lie in a highly conserved region, probably involved in cyclooxygenase catalysis. Sequence comparisons of mouse and sheep PGG/H synthase also provided information about the heme-binding site of the enzyme. The sheep HYPR sequence (residues 274-277), which had been proposed to form a portion of the distal heme-binding site, is not conserved in the mouse PGG/H synthase, suggesting that this region is not the distal heme-binding site. One sequence, TIWLREHNRV (residues 303-312 of the sheep enzyme), is very closely related to the sequence TLW(L)LREHNRL common to thyroid peroxidase and myeloperoxidase. The histidine in this latter sequence is the putative axial heme ligand of these peroxidases. We suggest that the histidine (His-309) of sheep PGG/H synthase sequence is the axial heme ligand of this enzyme.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5586
    Product Catalog Name:
    Anti-Cyclooxygenase-1 Antibody