Sodium Dodecyl Sulfate-polyacryamide Gel Electrophoresis Research Articles

Thermal Denaturation of Tilapia Myosin and Its Subunits as Affected by Constantly Increasing Temperature

Purified tilapia myosin was digested with α-chymotrypsin and purified to obtain heavy meromyosin (HMM) and light meromyosin (LMM). Biochemical properties of tilapia myosin, HMM, and LMM were characterized. Surface hydrophobicity (S(o) ) showed an increase for myosin and HMM between 30 and 40 °C and reached a plateau at 70 °C. LMM, in a small magnitude, also showed a continuous increase to 70 °C. Total sulfhydryl content (TSH) demonstrated that the SH residue content of HMM was nearly double that of LMM. Surface reactive sulfhydryl groups (SRSH) for myosin and HMM were relatively unchanged from 10 to 30 °C but increased from 30 to 50 °C. The exposure of buried hydrophobic and sulfhydryl groups of myosin and HMM increased as the myosin and HMM were constantly heated. However, the TSH and SRSH results indicated that the stability of LMM was likely due to its α-helix conformation. Reducing and nonreducing sodium dodecylsulfate-polyacryamide gel electrophoresis helped to understand the role of disulfide bonds in thermal aggregation of tilapia myosin, HMM, and LMM.

Rheological and Biochemical Characterization of Salmon Myosin as Affected by Constant Heating Rate

Purified Chinook salmon myosin was studied using sodium dodecylsulfate-polyacryamide gel electrophoresis and densitometric analysis to determine its purity (approximately 94%). Myosin subjected to a constant heating rate began to form aggregates at >24 °C as measured by turbidity at 320 nm. Conformational changes, as measured by surface hydrophobicity (S(o)), began at 18.5 °C and continued to increase up to 75 °C after which it decreased slightly. Total sulfhydryl (TSH) content remained steady from 18.5 to 50 °C after which point the TSH began to drop. Surface reactive sulfhydryl groups gradually increased as the temperature increased from 18.5 to 55 °C and then followed a similar trend as TSH decreased. Presumably disulfide bond started to be formed at around 50 to 55 °C. Differential scanning calorimetry showed 4 peaks, 3 endothermic (27.9, 36.0, 45.5 °C), and 1 exothermic (49.0 °C). Dynamic rheological measurements provided information concerning the gelation point of salmon myosin that was 31.1 °C as samples were heated at a rate of 2 °C/min.

Purification and Characterization of Canine Serum Ferritin-binding Proteins

Ferritin-binding protein (FBP) is known to interact with circulating ferritins in mammals. Canine FBPs were purified from canine serum by affinity chromatography and were identified as IgM, IgG, and IgA by immunoblotting with alkaline phosphatase-labeled antibodies to canine IgM, IgG, and IgA heavy chains. Following further purification by application to a Sephacryl S-300 column, canine FBPs were separated into 81.3- and 27.7-kDa bands by sodium dodecyl sulfate-polyacryamide gel electrophoresis, and the 81.3-kDa band reacted with the anti-canine IgM heavy chain antibody. Purified canine FBP bound to canine liver ferritin, but not to canine albumin and transferrin. FBP showed greater binding to the expressed bovine ferritin H-chain homopolymer than to the expressed bovine ferritin L-chain homopolymer. The binding of FBP with canine liver ferritin was dose-dependently inhibited by anti-rat liver ferritin antibody, and the anti-ferritin antibody dissociated the bound FBP in a dose-dependent manner, even after binding FBP with liver ferritin. The canine ferritin H subunit peptide fragment with amino acid residues 148-155 (NH(2)-GDHVTNLR-COOH) in its C-terminal region was recognized by FBP. These results indicate that canine serum FBPs are autoantibodies to ferritin (IgM, IgG, and IgA) and that anti-ferritin autoantibody (IgM) recognizes the C-terminal region of ferritin H subunit.

Production of a surfactant- and solvent-stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007

An extracellular serine protease with novel surfactant- and solvent-stable alkaliphilic properties was purified from the culture supernatant of Bacillus subtilis TKU007 with shrimp shell wastes as the sole carbon/nitrogen source. The TKU007 protease showed suppressing effect on the chitosanase, which appeared at the 1st day. The molecular mass of TKU007 protease determined by sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) and gel filtration was approximately 28 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, thermal stability, K m, and V max of TKU007 protease was 7–11, 50 °C, pH 5–11, 50 °C, 0.13 mg/mL, and 0.86 U/mL, respectively. More than 80% of its original activity was retained even after preincubation for 10 days at 25 °C in the presence of 25% tested organic solvents. Additionally, the TKU007 protease retained 100%, 100%, 50%, and 65% of its original activity in the presence of 2% Tween 20, 2% Tween 40, 2% Triton X-100, or 0.5 mM SDS, respectively. In conclusion, the novelties of the TKU007 protease include its high stability to the solvents, surfactants, and alkali. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.

Degradation and inactivation of photosystem I complexes during linear heating

The heat effect on photosytem I (PSI) was characterized by visible and fluorescence spectroscopy, oxygen electrode polarography, sodium dodecyl sulfate–polyacryamide gel electrophoresis (SDS–PAGE) and Fourier transform infrared (FTIR). During linear heating, the absorption maximum at 680 nm of PSI decreased and blue shifted, of which pigments and polypeptides in PSI complexes degraded at high temperature. The chlorophyll a (Chla) molecules with maximum absorption at 683 nm in the fourth derivative absorption spectra, which are mainly attributed to light-harvesting complex of PSI (LHCI), were more sensitive to high temperature. So it was suggested that Chla (absorbing at 683 nm) was destroyed firstly by elevated temperature. The characteristic peak at 728 nm and the ratio of F728–F720 to F680 in 77 K fluorescence spectra decreased apparently when temperature increased, showing that high temperature blocked energy transfer in LHCI and the energy transfer from LHCI 680 to LHCI 730 and PSI reaction center was inhibited. SDS–PAGE displayed that PsaA/B subunits and LHCI subunits were degraded and aggregated at different levels upon linear heat treatment. However, at high temperature especially at 90–100 °C, the PsaA/B subunits disappeared completely while LHCI subunits remained, indicating that the core complex subunits were more sensitive to heat treatment than LHCI subunits. The probable reason for the above results is that during linear heating the disconnection of the antenna from the reaction center occurred and then followed by the inhibition of the photochemical charge separation in reaction center. The oxygen uptake rate decreased with the increase of temperature, and it was almost completely lost at around 70 °C, demonstrating that the PSI complexes were destroyed seriously when the temperature reached 70 °C. FTIR analysis indicated that a major conformational transition of PSI complexes occurred above 60 °C, which was accompanied by unfolding of α-helical to the β-sheet structures.

Increase in electron transfer activity in photosystem II of spinach thylakoids caused by conversion of phosphatidylglycerol to phosphatidic acid molecules

The techniques of oxygen electrode polarography, sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) were employed to investigate the effect of phospholipase D treatment on physiological function of spinach thylakoids. It was shown that the phospholipase D treatment on thylakoid resulted in the degradation of phosphatidylglycerol (PG) and occurrence of phosphatidic acid (PA). The changes of PG to PA molecules caused an increase in oxygen evolution in photosystem II (PSII), which was accompanied by an uncoupling effect on thylakoid membrane. It was revealed that the head-groups of PG molecules play an important role in the maintenance of the appropriate physiological activity of thylakoid membrane.

Isolation and characterization of a Lactobacillus plantarum bacteriophage, ΦJL-1, from a cucumber fermentation

A virulent Lactobacillus plantarum bacteriophage, ΦJL-1, was isolated from a commercial cucumber fermentation. The phage was specific for two related strains of L. plantarum, BI7 and its mutant (deficient in malolactate fermenting ability) MU45, which have been evaluated as starter cultures for controlled cucumber fermentation and as biocontrol microorganisms for minimally processed vegetable products. The phage genome of ΦJL-1 was sequenced to reveal a linear, double-stranded DNA (36.7 kbp). Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) profiles indicated that ΦJL-1 contains six structural proteins (28, 34, 45, 50, 61, and 76 kDa). Electron microscopy revealed that the phage has an isometric head (59 nm in diameter), a long non-contractile tail (182 nm in length and 11 nm in width), and a complex base plate. The phage belongs to the Bradley group B1 or Siphoviridae family. One-step growth kinetics of the phage showed that the latent period was 35 min, the rise period was 40 min, and the average burst size was 22 phage particles/infected cell. Phage particles (90%) adsorbed to the host cells 20 min after infection. Calcium supplementation (up to 30 mM CaCl 2) in MRS media did not affect the first cycle of phage adsorption, but promoted rapid phage propagation and cell lysis in the infection cycle subsequent to adsorption. The D values of ΦJL-1 at pH 6.5 were estimated to be 2.7 min at 70 °C and 0.2 min at 80 °C by a thermal inactivation experiment. Knowledge of the properties of L. plantarum bacteriophage ΦJL-1 may be important for the development of controlled vegetable fermentations.

Ornithine Carbamoyltransferase from Spinacea oleracea: Purification and Characterization

Ornithine carbamoyltransferase (OCT) from spinach (Spinacea oleracea L.) was purified to homogeneity and studied for some kinetic and structural properties. The enzyme showed a specific activity of 436 U mg−1, its molecular mass was approximately 118 kDa as estimated by Sephacryl S-200 gel filtration chromatography, the purified protein ran as a single band of 38 kDa in sodium dodecyl sulfate-polyacryamide gel electrophoresis. The enzyme catalyses an ordered bi-bi-sequential reaction in which carbamoyl phosphate binds first, followed by L-ornithine; L-citrulline leaves first, followed by phosphate. The Michaelis constant was 0.19 mM for L-ornithine and 13.1 µM for carbamoyl phosphate; the dissociation constant for the enzyme and carbamoyl phosphate complex was of 19 µM. The Km of the reaction decreases from pH 6.0 to pH 10.4. The enzyme is heat-labile, but it was protected from thermal inactivation by substrates; more by ornithine alone than by two substrates acting together.

Production of activin-binding protein by rat granulosa cells in vitro

We have developed an assay method for activin-binding protein, which exploits its high affinity for sulfated polysaccharides. We used this method to investigate the production of activin-binding protein by rat ovarian granulosa cells, in vitro. The production of activin-binding protein by granulosa cells was dependent on the cell density; the maximum was observed at 6 × 10 5 cells/ml. Follicle-stimulating hormone (FSH), but not luteinizing hormone (LH), enhanced production significantly. Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) and ligand blotting analyses of the activin-binding protein secreted by rat granulosa cells demonstrated it was the same protein molecule as that purified from rat ovaries. It is inferred from these results that the granulosa cell is a source of ovarian activin-binding protein and that its secretion is regulated by FSH.

Purification and immunological properties of vanadate sensitive Mg: ATPase from plasma membrane of maize roots

The vanadate-sensitive ATPase from maize ( Zea mays L.) root plasma membrane was partially purified from KI-washed microsomes by discontinuous sucrose gradient centrifugation followed by deoxycholate (DOC) treatment, l-l-lysophosphatidyl-choline (lyso-PC) solubilization and glycerol gradient centrifugation. The plasma membrane preparation had a relatively high specific activity of ATPase activity (182 μmol P i/h (mg proteins). During lyso-PC solubilization, the presence of Mg:ATP and vanadate substantially improved the recovery of active ATPase. The 100-kDa polypeptide of the plasma membrane ATPase was isolated by sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) and used to raise a specific polyclonal antiservm. The addition of antiserum to the lyso-PC solubilized plasma membrane resulted in a loss of 90% of the total ATPase activity from the supernatant fraction after centrifugation. The antiserum also inhibited the proton transport activity of reconstituted plasma membrane vesicles. Western blot experiments showed that a single band of 100 kDa reacted with the anti-ATPase antibodies. Enzyme-linked immunosorbant assay (ELISA) analysis indicated that the antiserum exhibited specificity to plasma membrane as compared to other subcellular membranes. Thus, the antiserum obtained in this study is specific for the 100-kDa polypeptide of the plasma membrane ATPase.

Nucleotide sequence of the P1 attachment-protein gene of Mycoplasma pneumoniae

The specific attachment of Mycoplasma pneumoniae to the respiratory ciliated epithelium is mediated by a surface protein designated P1. The nucleotide (nt) sequence of the P1 attachment-protein gene has been determined and the amino acid (aa) sequence deduced. mRNA and cDNA sequencing confirm that this gene is transcribed in M. pneumoniae. The predicted amino acid sequence matches the N-terminal 12 aa residues of P1 protein from M. pneumoniae [Jacobs et al. J. Gen. Microbiol. 133 (1987) 2233–2236] beginning with Asn at aa position 60, where aa 1 represents the first codon of the open reading frame (ORF). Notably, the Trp at aa position 69 aligns with a UGA codon deduced from the nucleotide sequence, providing supporting evidence that UGA is read as Trp rather than stop in M. pneumoniae. Analysis of the first 59 aa suggests that it is probably a leader sequence that is processed to yield the mature protein. The codons of the mature P1 protein sequence represent 1568 aa with a calculated M r of 169758. A unique feature of this protein sequence is the lack of cysteine, and this was confirmed by sodium dodecyl sulfate-polyacryamide gel electrophoresis of M. pneumoniae proteins metabolically labeled with radioactive cysteine or methionine. This study has revealed that the 4881 nt of the P1 structural gene are flanked by ORFs, and there are no obvious ribosome-binding sites or transcription termination sequences in the immediately adjacent regions. This suggests that the P1 gene is transcribed as part of a larger polycistronic message. In addition, a number of untran scribed and therefore nonfunctional P1 epitope sequences were found in the M. pneumoniae genome; their purpose remains unknown.