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Objectives Bioactive peptides derived from receptor binding motifs of native proteins are a potent source of bioactive molecules that can induce signalling pathways. These peptides could substitute for osteogenesis promoting supplements. The work presented here compares three kinds of bioactive peptides derived from collagen III, bone morphogenetic protein 7 ( BMP-7) and BMP-2 with their potential osteogenic activity on the model of porcine mesenchymal stem cells ( pMSCs). Materials and methods pMSCs were cultured on electrospun polycaprolactone nanofibrous scaffolds with different concentrations of the bioactive peptides without addition of any osteogenic supplement. Analysis of pMSCs cultures included measurement of the metabolic activity and proliferation, immunofluorescence staining and also qPCR. Results Results showed no detrimental effect of the bioactive peptides to cultured pMSCs. Based on qPCR analysis, the bioactive peptides are specific for osteogenic differentiation with no detectable expression of collagen II. Our results further indicate that peptide derived from BMP-2 protein promoted the expression of mRNA for osteocalcin ( OCN) and collagen I significantly compared to control groups and also supported deposition of OCN as observed by immunostaining method. Conclusion The data suggest that bioactive peptide with an amino acid sequence of KIPKASSVPTELSAISTLYL derived from BMP-2 protein was the most potent for triggering osteogenic differentiation of pMSCs. [ABSTRACT FROM AUTHOR]

1-Pyreneisothiocyanate was shown to be an inhibitor of Na+/K+-ATPase. Reverse-phase HPLC and activity studies indicated binding of 1-pyreneisothiocyanate at the H4-H5 loop of the α subunit and competition with the fluorescein 5′-isothiocyanate for the E1ATP site. While fluorescein 5′-isothiocyanate, the fluorescent ATP pseudo-analog, was shown to be immobilized at the E1ATP site, there was no possibility to draw any conclusion about the flexibility of the E1ATP site due to its short lifetime. Employing 1pyreneisothiocyanate as a long-lived fluorophore and a label for the E1ATP site, we found that the ATP-binding site of Na+/K+-ATPase and, in fact, the whole large intracellularly exposed H4-H5 loop of the catalytic α subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function. [ABSTRACT FROM AUTHOR]

To probe the pH value in the microenvironment of the cardiac glycoside-binding site of Na+/K+- ATPase, pH-sensitive fluorescent derivatives of ouabain were synthesized. The fluoresceinyl derivative of ethylenediamino-ouabain CREDO) had a pKs of 6.0 and showed a H--dependent fluorescence change, when its ratio of excitation at 490 nm/450 nm was recorded at 530 nm. Binding of FEDO inactivated Na¯/K+-ATPase at 37°C and pH 7.25 in a slow time-dependent process under the conditions of backdoor phosphorylation with kon of 891 s-1 M-1. The complex dissociated with koff of 0.35×10-3 s-1 resulting in a Kd value of 0.4 μM for the FEDO · enzyme complex. Binding of FEDO was associated with a decrease of the excitatory fluorescence ratio at 490 nm/450 nm which could be used to convert this change into a pH value. A pH value of 5.1 ± 0.2 was calculated to exist in the microenvironment of the FEDO-enzyme complex. This pH value was independent of the pH of the incubation medium used to form the FEDO · enzyme complex. Analysis of the accessibility of the fluorophore in the FEDO · enzyme complex to the dynamic quencher potassium iodide detected a decrease of the Stern­Volmer constant from 6.2 mM-1 (free FEDO) to 1.5 mM-1 (FEDO · enzyme complex} indicating thereby a limited accessibility of the fluorophore to anions. Analysis of the microenvironment of the fluorescein residue of the FEDO · enzyme complex by measurements of the anisotropy and the fluorescence half-life time revealed that both processes differed significantly when H2O was replaced by D:O. We conclude, therefore, that a pH of 5.1 ± 0.2 exists in the vicinity of ouabain that is hidden in the depth of the receptor site when the ouabain, receptor complex has been formed. [ABSTRACT FROM AUTHOR]