Determination of binding parameters of cyclic AMP and its analogs to cyclic AMP-dependent protein kinase by the fluorescent probe method.

The method for determination of dissociation constants for cyclic AMP and its analogs bound to cyclic AMP-dependent protein kinase from pig brain is described. The technique for measuring the binding parameters of the ligands is based on the changes in the fluorescent spectrum of etheno cyclic AMP once it is bound to protein kinase. The dissociation constants for a number of nonfluorescent cyclic AMP analogs were determined in the competitive displacement of etheno cyclic AMP by these analogs. The number of cyclic AMP-binding sites in the pig brain protein kinase was found to be 2.2; no cooperativity was observed upon binding. The holoenzyme complex (Mr = 180,000) of the protein kinase under study was established to have the stoichiometry of R2C2 type under native conditions.     

Functional degradation of myelin basic protein. Proteomic approach

Proteolytic degradation of autoantigens is of prime importance in current biochemistry and immunology. The most fundamental issue in this field is the functional role of peptides produced when the specificity of hydrolysis changes during the shift from health to disease and from normal state to pathology. The identification of specific peptide fragments in many cases proposes the diagnostic and prognostic criterion in the pathology progression. The aim of this work is comparative study of the degradation peculiarities of one of the main neuroantigen, myelin basic protein by proteases, activated during progress of pathological demyelinating process, and by proteasome of different origin. The comparison of specificity of different studied biocatalysts gives reason to discuss the critical change in the set of myelin basic protein fragments capable to be presented by major histocompatibility complex class I during neurodegeneration, which can promote the progress of autoimmune pathological process.     

Recombinant Fragment of the Extracellular Domain of Human Desmoglein 3 Fused with the Fc-Fragment of Human IgG1 Selectively Adsorbs Autoreactive Antibodies from the Sera of Pemphigus Patients

Doklady Biochemistry and Biophysics - Using the recombinant second fragment of the extracellular domain (EC2) of human desmoglein type 3 (Dsg3) as an affinity ligand, an immunosorbent was obtained...     

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D₄K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective K_m values of 19 μM and 20 μM and k_cat of 115 and 92 s⁻¹. Fusion proteins containing a D₄K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.     

Chemical polysialylation: Design of conjugated human oxyntomodulin with a prolonged anorexic effect in vivo

Recombinant gut hormone oxyntomodulin (OXM) is known to act as a satiety signal in human subjects and has therapeutic potential as an appetite controlling agent. The only form of this hormone that has a prospective use is a modified one, because native OXM has a very short half-life in vivo. Conjugation of OXM and the natural hydrophilic polymer polysialic acid (PSA) may significantly improve its half-life. Chemical polysialylation in vitro was used to create a long-acting form of OXM, the polysialic acid–oxyntomodulin (PSA–OXM) conjugate. The conjugation site was identified using mass shift comparative analysis of Asp-N proteolytic digests. The anorexic effect of the conjugate was tested on the lean, fasted mouse model. A two-stage purification technique was developed to obtain a homogeneous PSA–OXM conjugate, suitable for in vivo testing. The N-terminal backbone primary amino group was found to be the only point of conjugation. The conjugate obtained was resistant to the DPP-IV protease. A single injection of PSA–OXM at 15 μmol/kg dose was sufficient to maintain a steady decrease in food consumption for 8 h (P < 0.05). The length of the anorexic effect achieved is comparable to other long-acting derivatives of OXM but it requires a much higher dose for administration. It is expected that site-directed attachment of the PSA chain to the inner residues of OXM, away from the site of interaction with receptors, would produce a compound with a higher specific activity but comparable stability in the bloodstream. The conjugation technique used may be used to create OXM derivatives and other related hormones to obtain long-lasting variants, with improved suitability for clinical use.     

A Structure–Function Study of a Catalytic Antiidiotypic Antibody to the Human Erythrocyte Acetyl Cholinesterase

The catalytic monoclonal antibody 9A8 (MA 9A8), antiidiotypic to the antibody AE-2 (MA AE2) produced to the active site of acetyl cholinesterase from human erythrocytes, was subjected to a structure–function study. The specific binding of MA 9A8 to MA AE2 (K 2.26 × 10⁹ M^–1) was found by the method of surface plasmon resonance, and the functional activity of MA 9A8 was demonstrated. Unlike acetyl cholinesterase, this antibody specifically reacted with the irreversible phosphonate inhibitors of esterases. A peptide map of MA 9A8 was analyzed by MALDI mass spectrometry. The Ser99 residue of its heavy chain was shown to be within the active site of the catalytic antibody. A computer modeling of the MA 9A8 active site suggested the existence of a catalytic dyad formed by Ser99 and His35. A comparison of the tertiary structures of the MA 9A8 and the 17E8 monoclonal antibody, which also exhibited the esterase activity and was produced to the stable analogue of the reaction transition state, indicated a practically complete coincidence of the structures of their presumed active sites.