Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.
Level of malonic dialdehyde, lipid peroxidation, and activities of main antioxidant enzymes were studied in respiratory-competent and respiratory-deficient strains of Saccharomyces cerevisiae yeast. The data confirm the hypothesis of V.P. Skulachev that the respiratory system is directly involved in the antioxidant defense of the cells.
Chemical modification of tryptophan residues with N-bromosuccinimide and their photooxidation in the presence of trichloroethanol inhibited the activity of adenosine deaminase purified from gray and white matter of calf brain. Only two of six modified residues are important for enzyme activity. Preliminary kinetic data indicate that these essential tryptophan residues are adjacent to the substrate-binding site of the enzyme.
The effects of ruthenium red, pH, and sodium ions on active and passive Ca2+ transport were studied in digitonin-treated (0.1 mg/ml) suspension of myometrial cells using radiolabeled 45Ca2+. The inhibitor of mitochondrial Ca2+ accumulation ruthenium red (10 microM) suppressed active accumulation of the cation in permeabilized myocytes by 78-85%. Ruthenium red-sensitive Ca2+ accumulation significantly (10-fold, mean) exceeds ruthenium red-resistant Mg2+, ATP-dependent Ca2+ accumulation assayed in the absence of oxalate and inhibited by 50 nM tapsigargin (blocker of endo/sarcoplasmic reticulum calcium pump). Acidification of incubation medium (pH 8.0-6.0, Tris-malcate/KOH buffer) inhibits ruthenium red-sensitive active Ca2+ accumulation in permeabilized myocytes and passive release of this cation in the dilution medium after blockage of mitochondrial Ca2+ accumulation by ruthenium red. Partial isotonic substitution of KCl (135-95 mM) with NaCl (15-55 mM) in incubation medium did not affect the initial rate of active ruthenium red-sensitive Ca2+ accumulation and the kinetics of passive Ca2+ release from permeabilized cells after blockade of active Ca2+ accumulation by ruthenium red. Mechanism of Ca2+ exchange in myometrial mitochondria is discussed.
Complete amino acid sequence of IT1 protease inhibitor and partial amino acid sequences of IT2 and IT4 protease inhibitors from buckwheat Fagopyrum esculentum Moench seeds were determined by automatic Edman degradation and mass spectrometry. IT1 inhibitor comprises 69 amino acid residues and its molecular mass is 7743.8 D. N-terminal 48 amino acid residues of IT2 inhibitor are identical to similar sequence of IT1 inhibitor. The sequence of 10 amino acid residues of IT4 inhibitor is completely identical to a part of the sequence of IT1 inhibitor C-terminally adjacent to its active site. Analysis of amino acid sequences of IT1, IT2 and IT4 inhibitors suggests that the proteins are the members of the potato proteinase inhibitor 1 family and include Arg-Asp residues in their active site.
Exposure of proteins on the surface of the small (30S) ribosomal subunit of Escherichia coli was studied by the hot tritium bombardment technique. Eight of 21 proteins of the 30 S subunit (S3, S8, S10, S12, S15, S16, S17, and S19) had virtually no groups exposed on the surface of the particle, i.e., they were mainly hidden inside. Seven proteins (S1, S4, S5, S7, S18, S20, and S21) were all well exposed on the surface of the particle, thus being outside proteins. The remaining proteins (S2, S6, S9 and/or S11, S13, and S14) were partially exposed. On the basis of these results a reconcilement of the three-dimensional protein map of the small ribosomal subunit has been done and corrected model is proposed.
The review discusses two related methods--counterflow isotachophoresis and immunoaffinity electrochromatography on porous membranes developed by the authors. The methods exploit the peculiarity of electroendosmosis in porous membranes at the conditions of discontinues electrophoresis. There are described the principles and application of the above methods for automatic performance of electrophoretic analysis of low-protein biological fluids, of western blotting, epitope analysis of monoclonal antibodies and multiantigen assays. The possibility of DNA-binding protein detection is also considered.
A novel footprinting method was recently developed which identifies phosphate groups of RNA involved in strong RNA-RNA and RNA-protein interactions. The method is based on iodine-dependent RNA cleavage at phosphothioate groups as long as these groups are not protected from iodine. Our recent studies of mRNA and tRNA regions protected in active ribosomes are summarized; initiation state of ribosomes as well as two elongation states in pre- and post-translocational states were analyzed. Only one phosphate group of mRNA, which was two positions upstream of the decoding codons, was weakly protected in longation complexes, whereas this group and the phosphate groups in the Shine-Dalgarno sequence were protected in the initiation complex. No protection was observed downstream of the decoding codons. On the contrary, numerous phosphate residues of tRNA were protected by the ribosome. The tRNA protection patterns significantly varied between two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA was not significantly affected by translocation. The data indicate that both tRNA molecules are tightly bound to the ribosome, whereas mRNA is fixed predominantly by two tRNAs via codon-anticodon interaction. A possible translocation mechanism is suggested.
Analysis of recent data on telomeres and telomerases, limitations of proliferative potential of untransformed cells (Hayflick's limit), kinetics and molecular mechanism of cell proliferation and differentiation, and factors that determine the longevity of multicellular organisms suggests that: (i) chromosome end shortening during consecutive divisions of cells that lack telomerase limits cell proliferation beyond Hayflick's limit; (ii) Hayflick's limit can be a manifestation of mechanisms that determine the size of the living organism (it correlates with longevity) but not the longevity of multicellular organisms.
An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis O10 and studied after full acid hydrolysis and carboxyl reduction by 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected heteronuclear 1H,13C multi-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY). It was found that the polysaccharide contains 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-galacturonic acid, and L-altruronic acid, and the following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: [sequence: see text]
Site-specific endonuclease R. AspMI was isolated and purified to apparent functional homogeneity from Acinetobacter species (strain M). The enzyme recognizes symmetrical DNA sequence 5'-AGG decreases CCT-3' and cleaves it at the site indicated by the arrow forming blind DNA ends. The endonuclease is an isoschizomer of the StuI endonuclease. Cleavage of the DNA site was inhibited by dcm-methylation. AspMI is approximately equal to 30 kD monomer.
Suggested method for calculation of solvent accessibility of tryptophan and tyrosine residues in water-organic mixtures is based on evaluation of second order derivatives of UV spectra. Second order derivative of the spectrum enable differentiation between direct effects of the organic solvent on spectral characteristics of exposed aromatic amino acid residues and the effect of the organic solvent on protein conformation. The method is suitable for calculation of exposition of tryptophan in water-organic mixtures if the content of tyrosine amino acid residues in protein does not exceed the content of tryptophan. Similar method of calculation of exposition of tyrosine residues can be used in proteins when the content of tyrosine is at least 5-fold higher than the content of tryptophan. For example, solvent accessibility of tryptophan residues of chymotrypsin is calculated in the presence of various organic solvents including ethanol, dioxane, and dimethyl formamide.
Conjugates of insulin with duck egg-white ovomucoid and soybean trypsin inhibitor were synthesized. The conjugates are highly stable to alpha-chymotrypsin treatment. Oral administration of insulin-ovomucoid conjugates in rabbits significantly lowered blood glucose level unlike administration of insulin-soybean trypsin conjugate.
Clinical modification of cytochrome P450scc with fluoresceine isothiocyanate (FITC) results in selective incorporation of the fluorescent label into Lys338. According to affinity chromatography on immobilized adrenodoxin, fluorescently labeled cytochrome P450scc can interact with ferredoxin. Fluorescent labeling did not significantly affect the enzymatic activity of the hemoprotein at the stage of the first electron transfer. Distance between the heme and labeled Lys338 (2.81 nm) was determined using the efficiency of the fluorescent resonance energy transfer within the donor-acceptor pair (FITC-heme); also changes in this distance were measured during the transitions of cytochrome P450scc from high to low spin state and from oligomeric to monomeric form as well as after its conversion to cytochrome P420 by temperature or alkaline treatment and after complete denaturation of the protein globule in 6 M guanidine chloride. Thus, chemical modification of cytochrome P450scc by selective labeling with FITC is informative method which can be used to monitor conformational changes in the cytochrome P450scc molecule during enzymatic catalysis.
Three main groups of chemicals influence melittin-induced hemolysis including neutral compounds and inhibitors and activators of hemolysis. Inhibitors include divalent cations Zn2+ and Ca2+, albumin, DIDS, etc.; their potency significantly increases if they are present at early stages of peptide-membrane interaction. The rate of melittin-induced hemolysis depends on time of preincubation with the cells in physiologic saline but does not depend on the presence of inhibitors or activators. Longer incubation increases the rate of hemolysis. These effects can be due to membrane inhibitory components with specific affinity to melittin which initially protect the membrane from its lytic effect; these components can dissociate from the cell surface after dilution and incubation in physiologic saline. According to the suggested model, characteristics of peptide-induced hemolysis of erythrocytes are determined by sequential stages of peptide-membrane interaction and depend on the formation of triple non-lytic complex comprising the membrane inhibitory component, the blocker, and the peptide; the complex inhibits destruction of the membrane.
Fragments of the ribosomal protein L1 from Thermus thermophilus were obtained by limited trypsinolysis and spontaneous proteolysis. Binding of the intact L1 and its proteolytic fragments to 23S ribosomal RNA was studied. First eight N-terminal amino acids are important for RNA binding because protein L1 lacking these amino acids exhibits reduced 23S rRNA binding. Additional cleavage of the polypeptide chain between residues 36 and 37 completely abolishes RNA binding. Comparison of these data with recently determined structure of protein L1 from T. thermophilus suggests the nature of interactions which can determine specific and strong association of the protein L1 with 23S rRNA.
The search of conserved motifs was performed in enzymes catalyzing acyladenylate formation using ATP as AMP-donor. Besides a known motif, we have found a second conserved motif. Screening the SWISS-PROT database for occurrence of the motifs have showed that both motifs are highly characteristic and occur in all proteins of this superfamily. The motifs are separated by 200-250 residues in all sequences. It may suggest that the both motifs belong to the structural unit involved in acyl adenylate formation.
The effect of cisplatin and the new drug cycloplatam (amine (cyclopentylamine)-S-(-)-malatoplatinum (II)) on protein kinase C (PKC) activity and Ca(2+)-dependent binding of PKC to T lymphocytes membranes was studied in vivo and in vitro. At first, the effect of the drugs on PKC activity of intact and activated lymphocytes was studied in vivo. In 48 hours after intraperitoneal injection of mice with therapeutic doses of the drugs, PKC activity of intact lymphocytes was differentially affected. Cisplatin did not practically alter the enzyme activity, whereas cycloplatam inhibited the PKC activity by 37% versus control. In lymphocytes activated by mouse P-388 leukemia cells in vivo, the drugs caused almost complete suppression of PKC activity and Ca(2+)-dependent binding of the enzyme to the membranes. The drugs were effective in intact cells. After incubation of intact lymphocytes in vitro for 24 hours with cisplatin or cycloplatam (10(-5)M), PKC activity was increased 1.62- and 1.35-fold, respectively, versus control. Ca(2+)-dependent binding of the enzyme to the membranes was also increased 1.61- and 1.36-fold by cisplatin and cycloplatam, respectively. On the contrary, at 10(-4) M concentration under similar conditions, the drugs did not affect the PKC activity of the lymphocytes. Furthermore, cycloplatam, unlike cisplatin, reduced the PKC binding to cellular membranes by 31%. The mechanisms of the drugs effects on PKC activity are suggested. The data indicate that increase or decrease of PKC activity induced by the drugs cause stimulation or depression of functional activity of T lymphocytes, respectively. Thus, the membrane-bound PKC can play the key role in initiation and development of immunomodulatory effects of cisplatin and cycloplatam.
Some aspects of formation and functioning of the cholesterol hydroxylase system were studied. A hybrid protein was synthesized in E. coli composed of the modified form of the (NADPH)adrenodoxin reductase precursor (N-terminal domain) and the shortened adrenodoxin precursor (C-terminal domain). The modified reductase precursor contained 12 extra amino acid residues at the N-terminus and the N-terminally shortened adrenodoxin precursor had 17 C-terminal amino acids of its targeting presequence. The hybrid reduced cytochrome P450scc in a reconstituted system. Thus, neither the extra 44 amino acids at the N-terminus of the reductase nor the 17 amino acid linker affected the interaction of the active sites in the hybrid protein. These modifications do not interfere with the binding of prosthetic groups and formation of the active sites of two enzymes in the E. coli cells. Modified N-terminal sequence of the hybrid does not affect its import into heterologous mitochondria.