SAMS Peptide

XPS and ToF-SIMS investigation of alpha-helical and beta-strand peptide adsorption onto SAMs.[Pubmed:19891457]

Langmuir. 2010 Mar 2;26(5):3423-32.

14-mer alpha-helix and 15-mer beta-strand oligopeptides composed of leucine (L) and lysine (K) were used to investigate peptide adsorption and orientation onto well-defined methyl and carboxylic acid-terminated self-assembled monolayer (SAM) surfaces with X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). XPS showed that both peptides reached monolayer thickness on both SAMs, but significantly higher solution concentrations were required to reach this coverage on the methyl SAMs. This shows that the peptides adsorb more strongly onto the carboxyl-terminated SAMs. The excess oxygen detected by XPS and the H(3)O(+) signal detected by ToF-SIMS for the SAMs with adsorbed peptides indicated that water molecules are associated with the adsorbed peptides, even under ultrahigh-vacuum conditions. Changes in the number of L and K fragments detected by ToF-SIMS indicate that the beta-strand oriented differently on the two SAMs. The L side chains were preferentially associated with the methyl-terminated SAM, and the K side chains were preferentially associated with the carboxyl SAM. In contrast, little change in the ToF-SIMS K/L ratio was observed for the alpha-helix peptide absorbed on the two SAMs, indicating that ToF-SIMS was not as sensitive to the orientation of the alpha-helix peptide.

Peptide-based SAMs that resist the adsorption of proteins.[Pubmed:18928285]

J Am Chem Soc. 2008 Nov 12;130(45):14952-3.

In this paper we present a modular approach for the fabrication of surfaces to characterize protein-protein interactions. The approach is based on azido peptides with an optimized sequence which are then thiol-functionalized using an alkynyl thiol and "click" chemistry. From these peptide thiols we fabricated SAMs on gold to evaluate the protein resistance, using surface plasmon resonance spectroscopy, toward streptavidin, bovin serum albumin (BSA), and fibronectin.

The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell?[Pubmed:9759505]

Annu Rev Biochem. 1998;67:821-55.

Mammalian AMP-activated protein kinase and yeast SNF1 protein kinase are the central components of kinase cascades that are highly conserved between animals, fungi, and plants. The AMP-activated protein kinase cascade acts as a metabolic sensor or "fuel gauge" that monitors cellular AMP and ATP levels because it is activated by increases in the AMP:ATP ratio. Once activated, the enzyme switches off ATP-consuming anabolic pathways and switches on ATP-producing catabolic pathways, such as fatty acid oxidation. The SNF1 complex in yeast is activated in response to the stress of glucose deprivation. In this case the intracellular signal or signals have not been identified; however, SNF1 activation is associated with depletion of ATP and elevation of AMP. The SNF1 complex acts primarily by inducing expression of genes required for catabolic pathways that generate glucose, probably by triggering phosphorylation of transcription factors. SNF1-related protein kinases in higher plants are likely to be involved in the response of plant cells to environmental and/or nutritional stress.

Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities.[Pubmed:2598924]

Eur J Biochem. 1989 Dec 8;186(1-2):129-36.

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine. 2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of 63 kDa, is labelled using [14C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [gamma 32P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity. 3. In the absence of AMP the purified kinase has apparent Km values for ATP and acetyl-CoA carboxylase of 86 microM and 1.9 microM respectively. AMP increases the Vmax 3-5-fold without a significant change in the Km for either protein or ATP substrates. 4. The response to AMP depends on the ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 microM. Studies with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations. 5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.

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 assay.[Pubmed:2574667]

Eur J Biochem. 1989 Dec 8;186(1-2):123-8.

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.