Modification of functional groups of the streptokinase molecule during photooxidation

Photochemical oxidation with methylene blue as photosensitizer results in the destruction of one histidine residue in the streptokinase molecule. This process is characterized by the rate constant corresponding to the modification of free L-histidine and results in partial inactivation of the protein. The rate of protein photo oxidation and photoinactivation is pH-dependent. As can be judged from the results of CD spectroscopy and gel chromatography, in weakly acidic (but not in weakly alkaline) media the reaction results in conformation changes of the streptokinase globule which affect the state of the protein tryptophanyl residue. It was found that the imidazole group destroyed during the photooxidation reaction is not essential either for the specific activity of streptokinase or for the formation of is stable complex with human plasminogen. The specificity of modification of the streptokinase histidine residue during the photooxidation reaction is discussed.     

Stability of 3' dangling ends on the core helix of AUGCAU at various Na+ concentrations.

Stability increments of 3' dangling ends on the core helices AUGCAU at various Na+ concentrations are reported. The results show that all 3' dangling ends except 3'U dangling at low Na+ concentrations can stabilize the helix and this stabilization is very sequence dependent.     

Nucleotide 2',3'-cyclic monophosphokinase from actinomycetes

Streptomyces nucleotide 3'-pyrophosphokinase does not only transfer the 5'-beta, gamma-pyrophosphoryl group of ATP, ATP 3'-pyrophosphate or dATP to a variety of nucleotides at the 3'-OH site, but also adds 2',3'-cyclic terminal monophosphate to some suitable nucleotides with the use of diadenosine 5',5'-polyphosphates (n = 3-5). Examples are pA greater than p, ppA greater than p, pG greater than p, CpG greater than p, etc.     

A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation

We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa and no apparent cofactor requirement. Addition of HGAD to purified neurointermediate pituitary PAM (peptidylglycine alpha-amidating monooxygenase, EC 1.14.17.3) increases the rate of formation of amide products by an order of magnitude. Sequential additions of PAM and HGAD gave results consistent with PAM first catalyzing the formation of an intermediate that is subsequently, in a separate reaction, converted by HGAD to the final amide product. Experiments with olefinic inactivators demonstrate that HGAD is not required for turnover-dependent inactivation of PAM and, correspondingly, that HGAD activity is not affected by inactivators of PAM. As expected, HGAD has no effect on the rate of PAM-catalyzed sulfoxidation, where a reaction analogous to that occurring during amidation of glycine-extended substrates is not possible. On the basis of these results, we propose that peptide C-terminal amidation in neurointermediate pituitary is a two-step process, with PAM first catalyzing the conversion of a glycine-extended peptide to the alpha-hydroxyglycine derivative, which is in turn converted to the final amide product by HGAD.     

Flagellin as an object for supramolecular engineering

A model of tertiary and quaternary structure of E. coli flagellin is suggested. According to this model, the molecule consists of two independent parts. One of them is formed by the N- and C-terminal regions of the polypeptide chain and is responsible for polymerization properties. The other consists of the central region of the polypeptide chain and composes that part of the molecular globule that forms the flagella surface. It is suggested that different artificial supramolecular structures can be constructed from flagellin molecules by varying the surface domain, i.e. the central part of the polypeptide chain, and by using the domain responsible for polymerization properties as a universal block for assembly. The simplest possible artificial supramolecular structures that can be constructed from flagellins of different strains are discussed.     

Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation.

The human insulinlike growth factor I receptor was overexpressed in NIH 3T3 cells as well as human and rat primary fibroblast strains. The NIH 3T3 cells displayed a ligand-dependent, highly transformed phenotype. When exposed to insulinlike growth factor I or supraphysiologic levels of insulin, NIH 3T3 cells that expressed high levels of receptors formed aggregates in tissue culture dishes, colonies in soft agar, and tumors in nude mice. Expression of 1 million receptors per cell, a 40-fold increase above the base-line level, was required for anchorage-independent growth. Primary fibroblasts that expressed high levels of receptors displayed a ligand-dependent change in morphology and an increase in saturation density but did not acquire a fully transformed phenotype. The results demonstrate that when amplified, this ubiquitous growth factor receptor behaves like an oncogenic protein and is capable of promoting neoplastic growth in vivo.