PAG Electrophoresis Research Articles

Рекомбинантный химозин Camelus dromedarius в экспрессионной системе Pichia pastoris: очистка и анализ ферментативных свойств

Natural chymosin production is an expensive and complex process associated with ethical issues. The article introduces recombinant chymosin Camelus dromedarius (rChn-Cam) isolated from a P. pastoris expression system and optimized for different nutrient media at different zeocin concentrations. The sequence of prochymosin gene was obtained from NCBI BLAST. GS115/his4 P. prastoris served as a producer strain. The pPICZ(alpha) B vector with the AOXI promoter made it possible to construct the expression cassette. The experiment involved methods of genetical engineering and strain cultivation. The recombinant His-Tag-labelled proteins were isolated by the method of metal-affinity chromatography and analyzed using PAG electrophoresis and Western-blot analysis. The molecular weight was determined by MALDI-TOF MS while the concentration was defined spectrophotometrically. The shuttle expression plasmid pPICZ(alpha)B/proCYM_camel_pp_IDT revealed that the cell mass expansion of P. pastoris GS115/his4 (Mut+) should be performed with a preliminary introduction of 0.5% methanol. After the transformation of P. pastoris GS115/his4 and obtaining a strain-producer of P. pastoris/pPICZ(alpha)B/proCYM_camel_pp_IDT, the rate of cell mass gain started to correlate with the zeocin concentrations in two different media. Medium YPD was not fornified and contained 50, 100, and 200 μg/mL zeocin. MediumYPD was fortified with 0.00004% biotin and 1% glycerol and included 50, 100, and 200 μg/mL zeocin. The strain-producer grew better in the medium with a zeocin concentration of 50 μg/mL. The mass of rChn-Cam was 35.673 kDa after isolation and purification. When the pH of the substrate rose from 5.0 to 6.5, the coagulation activity decreased by 24%. The thermal inactivation threshold of rChn-Cam was 40–45°C. The unit of coagulation activity decreased as the zeocin concentration went up. The rChn-Cam concentration was in inverse correlation with the substrate coagulation time. In this research, the rChn-Cam obtained in the expression system of P. prastoris proved to be a good alternative to rChn used in the cheese industry.

The interplay between copper(II), human serum albumin, fatty acids, and carbonylating agent interferes with Cys 34 thiol reactivity and copper binding.

Cys34 thiol group of human serum albumin (HSA) represents major plasma antioxidant. Its reactivity is influenced by multiple factors. The influence of fatty acids (FA; saturated, mono, and poly unsaturated acids from fish oil) binding to HSA, on copper(II) binding affinity and Cys34 thiol group accessibility/reactivity, in the presence of carbonylation agent (methylglyoxal, MG) was examined. HSA-copper(II) content, thiol group reactivity, and HSA carbonylation level were monitored spectrophotometrically. Changes in HSA were followed by fluorescence spectroscopy and native PAG electrophoresis. FA/HSA molar ratio was screened by GC. Together, binding of copper(II) ions and FA to HSA increase the reactivity of Cys34 thiol group (depending on the type of FA), with constant contribution of copper(II) ions of one-third. Carbonylation of FA-HSA-Cu(II) complexes caused a decrease in the Cys34 thiol group content, accompanied by a decrease in the content of HSA-bound copper. The carbonylation level of guanidine groups was not affected by FAs and copper(II) binding. Fluorescent emission spectra of FA-HSA-Cu(II)-MG complexes showed conformational changes in HSA molecule. Although binding of fatty acids and copper ions caused a significant increase in the thiol group reactivity, Cys34 thiol from FA-HSA-Cu(II) complexes reacted with MG in smaller extent than expected, probably as a consequence of conformational changes introduced by carbonylation. Increase in the percentage of reacted-free thiol groups with MG (due to FA and copper binding) may not seem to be very significant, but it is very important in complex biological systems, where catalytic metal is present.

Intermolecular interactions in solutions of serum albumin

The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis, we have shown that there is dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0—1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M), the sizes of aggregates decreased, while higher urea concentrations induced formation of larger aggregates due to the unfolding of the protein.

Fatty acids binding to human serum albumin: Changes of reactivity and glycation level of Cysteine-34 free thiol group with methylglyoxal

Fatty acids (FAs) binding to human serum albumin (HSA) could lead to the changes of Cys-34 thiol group accessibility and reactivity, i.e. its scavenger capacity and antioxidant property. The influence of saturated, mono and poly unsaturated, and fish oil FAs binding to HSA on the carbonylation level and the reactivity of HSA-SH and HSA modified with methylglyoxal (MG-HSA-SH) was investigated. Changes of thiol group reactivity were followed by determination of pseudo first order rate constant (k′) for thiols reaction with 5,5′-dithiobis(2-nitrobenzoic acid). HSA changes were monitored using native PAG electrophoresis and fluorescence spectroscopy. For FA/HSA molar ratios screening, qTLC and GC were used. FAs increase thiol group carbonylation levels from 8% to 20%. The k′ values obtained for FAs-free HSA-SH and FAs-free MG-HSA-SH are almost equal (7.5×10−3 and 7.7×10−3s−1, resp.). Binding of all FAs amplify the reactivity (k′ values from 14.6×10−3 to 26.0×10−3s−1) of HSA-SH group for 2–3.5times in the order: palmitic, docosahexaenoic, fish oil extract, stearic, oleic, myristic and eicosapentaenoic acid, due to HSA conformational changes. FAs-bound MG-HSA-SH samples follow that pattern, but their k′ values (from 9.8×10−3 to 14.3×10−3s−1) were lower compared to unmodified HSA due to additional conformation changes of HSA molecules during carbonylation. Carbonylation level and reactivity of Cys34 thiol group of unmodified and carbonylated HSA depend on type of FAs bound to HSA, which implies the possibility for modulation of -SH reactivity (scavenger capacity and antioxidant property) by FAs as a supplement.

Properties of low-fat ultra-filtered cheeses produced with probiotic bacteria

Probiotics are live microorganisms that in certain numbers may confer a health benefit on the host. Nowadays, there are many dairy products on the market, especially fermented milks, with probiotics, and their popularity is rising. The aim of this article was to investigate the viability of commercial probiotic bacteria (Lactobacillus acidophilus LAFTI?L10 i Bifidobacterium lactis LAFTI?B94, DSM, Netherland) as well as their influence on the changes of composition, pH, proteolysis, microbiological status and sensory properties of low-fat ultra-filtered (UF) cheeses within 2 months of ripening. Low-fat cast ultra-filtered (UF) cheeses were produced according to the defined production procedure by mixing UF milk protein powder, skim milk and cream, without (control cheese A) and with adjunct probiotic culture (cheese B). The compositional parameters (milk fat, proteins and dry-matter content), pH, proteolysis parameters (water soluble nitrogen, nitrogen soluble in 5% PTA, urea and SDS PAG electrophoresis), as well as the numbers of starters and probiotic bacteria, were determined during ripening. In addition, sensory evaluations of cheeses were performed throughout the ripening time. A significant influence of probiotic strains on the composition, pH and primary proteolysis of cheese during ripening was not found. The counts of commercial probiotic bacteria were maintained at high levels (>107 cfug-1) during the overall ripening period, as a prerequisite of their therapeutic effects. The adjunct probiotic cultures enhanced the rate of secondary proteolysis, which was shown by the significantly higher levels of PTAN/TN of experimental compared to the control cheeses. The sensory evaluation showed that the overall aroma of low-fat cheeses was remarkably improved by the addition of the probiotic cultures used. Based on the results it can be concluded that the low-fat UF cheeses differ in good dietetic and functional properties as well as very acceptable sensory properties, and can be used as carriers of probiotics.

Role of bacteriochlorophyll in stabilization of the structure of the core and peripheral light-harvesting complexes from purple photosynthetic bacteria

Pheophytinization of bacteriochlorophyll (BChl) at low pH was investigated in the core (LH1) and peripheral (LH2) light-harvesting complexes, as well as in the ensemble of the reaction center (RC) with the LH1 complex. The stages in disintegration of the native BChl forms in the LH1 complex and in its ensemble with RC were revealed. They were observed as emergence of the absorption band of monomeric BChl and an increase in its intensity, followed by its transformation into the band of monomeric bacteriopheophytin (BPh) and then into the band of aggregated BPh. Unlike the LH1 complex, in the case of the LH2 complex monomeric BChl was never detected as an intermediate product. While the spectra revealed formation of monomeric BPh, its accumulation did not occur, since its aggregation is very rapid compared to that in the LH1 complex and in the RC-LH1 ensemble. PAG electrophoresis revealed that pheophytinization of BChl in the LH2 complex was accompanied by disruption of the stable cylindrical structure of this complex with emergence of characteristic fragments consisting of α and β peptides and bearing monomeric BPh, as well as of the α peptide aggregates bearing BPh aggregates. Unlike the LH2 complex, BChl pheophytinization in the LH1 complex did not result in its fragmentation. This is an indication of different types of structural stabilization in the LH1 and LH2 complexes. In the LH2 complex, coordination of bacteriochlorophyll Mg2+ by conservative histidine residues of the α and β polypeptides is the main factor responsible for the maintenance of its cylindrical structure. Stability of the LH1 complex is probably based primarily on the highly specific hydrophobic interactions between the surfaces of individual polypeptide chains, since the presence of hydrogen bonds results in autonomy of each αβ3BChl2 subunit, rather than in stabilization of the LH1 complex as a whole.

Cloning, expression, isolation, and properties of thymidine kinase from herpes simplex virus type 1, strain L2

A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.

Structural and functional changes in complement protein C4 under UV irradiation

The influence of ultraviolet (UV) light on the structural and functional states of the complement factor C4 was investigated using hemolytic and acid-base titration, PAG electrophoresis, and IR and UV spectrophotometry. UV doses of 75.5 and 755 J/m2 initiated C4 activation through changes in the globule structure (increased number of aromatic amino acids and ionogenic groups at the surface). The maximal dose of 2265 J/m2 has a destructive effect and decreases its C4 activity in the cascade of hemolytic reactions of the complement system.

Molecular characterization of glutenin alleles at the Glu-Dl locus

It is well known that the composition of high-molecular-weight (HMW) glutenin subunits impacts the bread making quality. The HMW subunits 1Dx5-1Dyl0 are typically associated with high dough strength and good bread making quality contrary to 1Dx2-lDyl2 subunits. Bread wheat cultivars from Institute of Field and Vegetable Crops in Novi Sad have been screened for the alleles present at Glu-Dl locus using traditional SDS PAG electrophoresis method and a new PCR based approach. The Glu-Dl locus was screened for two main x-type alleles which code for HMW glutenins 2 and 5, and two main y-type alleles which code for HMW glutenin subunits 10 and 12. Among the analyzed cultivars 55.6% expressed the presence of 1Dx5 and 1Dy10 alleles at the Glu-Dl locus. These results confirmed that by using marker-assisted selection (MAS) it is possible to identify genotypes with alleles for good bread making quality which could be successfully used in wheat breeding programs.

A novel Cu,Zn superoxide dismutase from the fungal strain Humicola lutea 110: isolation and physico–chemical characterization

The fungal strain Humicola lutea 110 produces a mangan- and a copper zinc-containing superoxide dismutases (SOD). In this study, the purification, N-terminal sequence and spectroscopic properties of the new Cu,Zn SOD are described. The preparation of the pure metalloenzyme was achieved via treatment of the strain with acetone followed by gel and ion exchange chromatography. The protein consists of 302 amino acid residues and has a molecular mass of approximately 32 kDa, as determined by PAG electrophoresis and 3100 U mg −1 protein-specific activity. It is a dimeric enzyme with two identical subunits of 15 950 Da, as indicated by SDS-PAGE, mass spectroscopic and amino acid analysis. The N-terminal sequence analysis of the Cu,Zn SOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Conformational stability and reversibility of unfolding of the dimeric enzyme were determined by fluorescence and circular dichroism (CD) spectroscopy. The critical temperature of deviation from linearity ( T c) of the Arrhenius plot ln ( Q −1−1) vs. 1/ T was calculated to be 68°C and the respective activation energy for the thermal deactivation of the excited indole chromophores is 42 kcal mol −1. The melting temperatures ( T m) were determined by CD measurements to be 69°C for the holo- and 61°C for the apo- enzyme. The fluorescence emission of the Cu,Zn SOD is dominated by ‘buried’ tryptophyl chromophores. Removal of the copper–dioxygen system from the active site caused a 4-fold increase of the fluorescence quantum yield and a 10 nm shift of the emission maximum position towards higher wavelength.

The Two-Stage Process of the Heat Shock Protein 90 Thermal Denaturation: Effect of Calcium and Magnesium

Scanning microcalorimetry, native PAG electrophoresis, and circular dichroism were used to characterize thermal denaturation and oligomerization of heat shock protein 90 (hsp90) and the calcium and magnesium effect on these processes. The calorimetric curve of the hsp90 dimer consists of two transitions centered at 53.8 and 63.1°C. Using specific ligand geldanamycin, we have found that N-terminal domains in the hsp90 dimer are melted independently in the lower-temperature peak, while the higher-temperature one comprises unfolding of two non-interacting parts of the middle domains and dimerization region. Unfolding of the N-terminal domain gives start to oligomerization of dimers; oligomers consist of dimers not dissociating upon denaturation. Calcium and magnesium strongly decrease the hsp90 thermostability and thereby cause oligomerization at lower temperature. We suggest that calcium affects the hsp90 oligomerization, known to be important for its chaperone activity, by shifting the unfolding temperature of the hsp90 N-terminal domain close to the heat shock temperature range.

Growth inhibitor effects on protoplasmic drought tolerance and protein synthesis in leaf cells of the resurrection grass, Sporobolus stapfianus

Hydrated leaves of the resurrection grass S.stapfianus Gandoger are not desiccation tolerant, but tolerance can be induced in them by moderate to severe drought stress. When brassinolide (BR) and methyljasmonic acid (MJA) were applied separately, each improved PDT by approximately 6 MPa. Exogenous abscisic acid (ABA) improved the protoplasmic drought tolerance (PDT) of suspended cells from hydrated leaves of S. stapfianus only slightly (about 1 MPa). BR, MJA or ABA treatment of leaves on fully hydrated S. stapfianus plants induced changes in the leaf protein complement (partitioned by 2-D PAG electrophoresis), with the induction of apparently novel proteins and increased and decreased abundances of other proteins. Most of the changes that were induced by MJA differed from those produced by ABA and also by BR. Two proteins increased in abundance after treatment of leaves with MJA, BR or ABA.

Sequence variation of allele 27 at the D1S80 locus.

In 180 unrelated Japanese individuals 18 examples of allele 27 were detected at the locus D1S80 (MCT118). On 6% polylacrylamide gels 5 out of these 18 alleles were found to migrate between allele 26 and allele 27, but closer to allele 27, and thus were labelled variants of allele 27. All 18 examples of allele 27 were sequenced and the results were compared. Although all had the same number of base pairs (578 bp) the five variants could be subdivided into three types. V1, V2 and V3. The variants and the standard allele were composed of the same kinds of repeat units, but the order of arrangement was different. We investigated whether it was possible to distinguish the standard allele 27, and the variants V1, V2, and V3 by PCR-RFLP. EcoRII and MspI which have restriction sites within the repeat units were adopted as restriction enzymes. The variants could be discriminated from each other after treatment of the PCR fragments with EcoRII or MspI, followed by PAG electrophoresis.

Population study of the STRs HUMCD4 and HUMF13A1 in Catalonia (Northeast Spain).

Allele and genotype frequencies were determined in a population sample from Catalonia (northeast Spain) for two short tandem repeat loci (HUMCD4 and HUMF13A1), using the polymerase chain reaction (PCR). After denaturing PAG electrophoresis, 6 alleles were identified for HUMCD4 in a sample of 157 unrelated individuals, and 11 alleles for HUMF13A1 in a sample of 141 individuals. No deviation from Hardy-Weinberg equilibrium was found. The HUMCD4 and HUMF13A1 loci demonstrated a heterozygosity of 0.6815 and 0.7305 respectively.

Population study of the STRs HUMTH01 (including a new variant) and HUMVWA31A in Catalonia (northeast Spain).

Allele and genotype frequencies for 2 short tandem repeat loci were determined in a population sample from Catalonia (Spain) using the polymerase chain reaction. After denaturing PAG electrophoresis, seven common and one variant (13.3) alleles were identified for HUMTH01 in a sample of 234 unrelated individuals, and seven alleles were found for HUMVWA31A in 162 individuals. No deviation from Hardy-Weinberg equilibrium was found. The observed heterozygosities are 76.92 and 79.62 respectively. The discrimination power determined for the individual loci is 0.928 and 0.937 respectively.

Properties of Isochorismate Hydroxymutase from Flavobacterium K3-15

Isochorismate hydroxymutase (isochorismate synthase, E.C. 5.4.99.6) catalyzes the interconversion of chorismic acid [1] and isochorismic acid [2]. The enzyme was extracted from a Flavobacterium K3-15 that overproduces vitamin K2 (i.e., menaquinones) and was purified to homogeneity. The N-terminal amino acid sequence and the mol wt (36,240 +/- 100 daltons) were determined by ms following SDS PAG electrophoresis. The enzyme was characterized (stability, cofactor requirement, isoelectrical point), and antibodies were raised which showed no cross reactivity with isochorismate hydroxymutase from Escherichia coli and Enterobacter aerogenes 62-1. The kinetic data of the enzyme are different from those observed for the corresponding enzyme from Escherichia coli and Galium mollugo.

動脈組織間液の血清リポ蛋白に関する研究

The interstitial fluid of arterial tissue constitutes a milieu interior for the intimal smooth muscle cells. Atherosclerotic foam cells arise from these cells when they are exposed to a high concentration of interstitial LDL. However, the available data on the concentrations of interstitial lipoproteins including LDL in normal arteries is unequivocal.In this study, the author has demonstrated the localization, form, and concentration of interstitial lipoproteins in terms of their apoproteins of A-I, A-II, B, C-II, C-III, and E in the normal intimas from 26 thoracic aortas and 19 pulmonary arteries of 45 autopsied cases (male 23, female 22, age 54±19).The localization of apoproteins was observed in the normal Intima of thoracic aorta and pulmonary artery by immunofluorescence: apo B localized mainly around the cell in the intima and along the internal elastic lamina, while apo A-I and A-II grouped in an islet form deep in the media. Apo C-II, C-III and E were sparsely present both in the intima and media. SDS-gradient PAG electrophoresis revealed all serum apoprotein bands with a _mobility corresponding to the serum counterparts.PAG disk electrophoresis showed that apoproteins were complexed with sudanophillic lipids in the interstitial fluid, and migrated in 3 bands of mobility corresponding to those of serum VLDL, LDL and HDL. Three dimensional form of recovered lipid-apoprotein complex was confirmed to be spheres of a diameter of about 500Å and 200Å by transmission and scanning electronmicroscopy.The concentrations of interstitial lipoproteins were measured by SRID for both thoracic and pulmonary interstitial fluids. Apo A-I was 5.2mg/ dl in thoracic aorta, while 0.4mg/dl in pulmonary artery, A-II 1.4mg/dl vs. 0.9mg/dl, B 18.2mg/dl vs. 10.3mg/dl, C-II 0.2mg/dl vs. 0.2mg/dl, C-III 1.1mg/dl vs. 0.4mg/dl and E 2.1mg/dl vs. 0.6mg/ dl, respectively. The concentrations of apoproteins gained in wet tissue weight were converted to the unit of mg/dl by using factors for water content of thoracic aorta 77±10%, and for pulmonary artery 81±19%, and the volume of extracellular space as 52±10% and 68±11%, respectively.These indicated that there exists a distinct difference in the concentrations of lipoproteins between serum and interstitial fluid of both thoracic and pulmonary intimas: interstitial LDL is 1/5 to 1/10 of serum counterpart, VLDL 1/5 to 1/7, and HDL 1/25 to 1/35, respectively. These lipoprotein levels maintained as a millieu interior by an endothelial barrier provides intimal cells with cholesterol necessary but sufficient for their cellular metabolism. The alteration in this milieu interior brings forth the accumulation of cholesterol in the cytoplasma of intimal cells, and leads to the formation of foam cells in the intima.

Selective Inhibition of DNA Chain Elongation Catalyzed by DNA Polymerases

Abstract The results of series of works on the properties of a large number of nucleoside 5′-triphosphates analogs in the reaction catalyzed by several DNA polymerases are summarized. Molecular mechanisms of substrate selection by DNA polymerases are not studied in detail. Therefore we have undertaken a comparative analysis of DNA polymerases from different sources employing nucleoside 5′-triphosphate analogs capable of incorporating into DNA chains terminating these chains elongation. Synthesis of a large line of nucleoside 5′-triphosphate analogs with substitution at the sugar residue has been performed. DNA polymerases have been isolated, and the synthesis of DNA has been studied using phage M13 DNA or phage MS2 RNA with synthetic deoxyoligonucleoti-de primers. The molecular mechanism of the substrate action has been determined by PAG electrophoresis of the reaction

前立腺特異抗原γ‐Ｓｅｍｉｎｏｐｒｏｔｅｉｎに関する研究

A relatively longer precipitin line in γ-globulin region was found in immunoelectrophoresis of seminal fluid using antiserum and this specific protein present in the seminal fluid was called as γ-seminoprotein (γ-Sm).γ-Sm was isolated and purified from seminal fluid by gel-filtration and isoelectrofocusing, and chemical property and γ-Sm level in seminal fluid were analysed.The results were;1. γ-Sm formed 5 to 6 bands in PAG electrophoresis.2. γ-Sm level in 15 seminal fluid samples was 500 to 1, 000mg/dl.3. Primary structure of γ-Sm was found to be consisted with 237 amino acid residues and possessed N-acetylglucosaminyl-asparagine linkage at 45 position.4. γ-Sm is one of serine proteases possessing active serine at 189 position from N-terminal.5. γ-Sm is an useful marker of seminal fluid in forensic medicine and also as a tumor marker for prostate cancer.6. γ-Sm was observed to be localized in prostatic cells by PAP method and the substance was demonstrated in perinuclear space, rough endoplasmic reticulum, Golgi apparatus, secretory granules and lysosome of prostatic epithelial cell by immunoelectron microscopic method.

健常人における血清コリンエステラーゼＣ５変異の推定頻度

We examined the distribution of C5 variant among 310 sera from healthy individuals. The frequency of the C5 variant was 2.3% and was similar to that in all Negro and Korea samples. The ChE activity for the C5 variant among healthy individuals was from 458 to 710U/l. Three cases of them showed lower ChE activity than 573U/l (mean+1SD of C5-individuals). These results suggest that we must try to detect the C5 variant not only in high ChE activity sera but also in low ChE activity sera. We caluculated the ratio of ChE activity to Alb (ChE/Alb) and then tried to detect the existence of C5 variant among the samples. However, it was difficult to detect the existence of the C5 variant completely. At present, our simple ChE activity staining method following PAG electrophoresis is most useful for detecting the C5 variant.