Preparative isoelectric focusing in ampholine electrofocusing columns versus immobiline polyacrylamide gel for the purification of biologically active leukoregulin

Preparative vertical and rotating horizontal (Rotofor) ampholine column and immobiline flat bed polyacrylamide gel isoelectric focusing were evaluated for the isolation of the biologically active acidic form of leukoregulin, a 50,000-Da glycoprotein lymphokine with tumor growth inhibitory activity. Leukoregulin secreted by normal human lymphocytes was concentrated by 10,000 nominal molecular weight size exclusion ultrafiltration and by DEAE anion exchange chromatography using step elution with 0.02 M Tris-HCl: 0.1 M NaCl, pH 7.4. Preparative isoelectric focusing was carried out in a 110-ml vertical column containing 1% ampholines in a pH 4-6 gradient at 15 W constant power for 16-18 h, in a Rotofor 55-ml horizontal column containing 2% ampholines in a pH 4-6 gradient at 12 W constant power for 4-6 h, or in an immobiline pH 4.5-6.5 gradient within a 5% polyacrylamide 120 X 110 X 5-mm flat bed gel at 3 W constant power for 16-18 h. Recovery of biologically active leukoregulin from the vertical and horizontal ampholine columns was similar. The pH 4.9-5.2 fractions from the Rotofor ampholine column contained 4-7% and the fractions from the immobiline gel contained 4% of the leukoregulin activity applied to the electrofocusing column or gel, respectively. Analytical immobiline isoelectric focusing of the leukoregulin in the pH 4.9-5.2 fractions from the Rotofor column demonstrated that a single silver staining band with a pI of 5.1 can be obtained by this rapid method of preparative isoelectric focusing.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

Studies on site directed mutant pig citrate synthases

The DNAs encoding the non-mutant and mutant forms of pig citrate synthase (PCS) were subcloned into an expression system to determine their synthesis and stability in E. coli gltA- cells that are defective in bacterial citrate synthase. GltA- cells that expressed the non-mutant PCS DNA grew on defined minimal acetate media and produced a constant level of PCS (0.43 U/mg protein). In contrast, when the gltA- cells were transformed with the DNA encoding PCS mutations in His274 or Asp375 the cells did not grow on minimal acetate media. The presence of the mutant PCS proteins in E. coli was confirmed by protein blot and immunoisolation analyses using an antibody specific for porcine heart citrate synthase. The activities of the mutant PCS enzymes were two orders of magnitude less than the non-mutant enzyme in the total cell lysates. The data indicate that the active site amino acids, His274 and Asp375, are essential for the catalysis activity of citrate synthase.     

Agents affecting adenylate cyclase activity modulate the stimulatory action of 1,25-dihydroxyvitamin D3 on the production of osteocalcin by human bone cells

The stimulation of osteocalcin synthesis by human osteoblast-like cells in response to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is antagonised by several bone regulatory agents. We have shown that agents which activate adenylate cyclase inhibit this action of 1,25(OH)2D3 on human osteoblast-like cells. Activation of adenylate cyclase, either via the stimulatory GTP-binding protein using cholera toxin, or directly at the catalytic via the stimulatory GTP-binding protein using cholera toxin, or directly at the catalytic subunit using forskolin, results in a suppression of osteocalcin synthesis. Whilst the activation of adenylate cyclase induces this inhibitory response, neither exogenous dibutyryl cyclic AMP nor the phosphodiesterase inhibitor, IBMX, exerted any apparent effect on the production of osteocalcin. The tumour promoting phorbol ester, 4 beta-phorbol 12,13-dibutyrate, also inhibited 1,25(OH)2D3-stimulated osteocalcin production. This was not apparent in response to the non-tumour promoting phorbol ester 4 beta-phorbol suggesting the involvement of protein kinase C.     

Role of cationic residues in cytolytic activity: modification of lysine residues in the cardiotoxin from Naja nigricollis venom and correlation between cytolytic and antiplatelet activity

Cardiotoxins and postsynaptic neurotoxins from snake venoms have similar primary, secondary, and tertiary structures. Cardiotoxins, however, in contrast to neurotoxins, exhibit general cytotoxicity. Comparison of the distribution of hydrophobic and charged amino acid residues in the three-dimensional structures of lytic cardiotoxins and nonlytic neurotoxins indicates the presence of a cationic site associated with a hydrophobic surface in cardiotoxins, but not in neurotoxins. A cationic site flanked by a hydrophobic site is a common structural feature shared by a wide variety of unrelated cytolysins and is predicted to determine the lytic activity of a large group of cytolysins. To determine the essential nature of the cationic site in cardiotoxin CTX-1 from Naja nigricollis crawshawii venom, we modified the positive charges of nine Lys residues to negative, neutral, or positive charges by succinylation, carbamylation, or guanidination, respectively. Circular dichroism studies indicated that these modifications did not affect the conformation of the cardiotoxin. Binding of the modified cardiotoxins to phospholipids was demonstrated by changes in the intrinsic fluorescence of native and modified CTX-1 after binding to phospholipid vesicles, and by resonance energy transfer with anthracene-phospholipid vesicles. Phospholipid binding was not affected by these modifications, but their binding preference was determined by the electrostatic interactions between the polypeptide and phospholipid. Both positively charged native and guanidinated CTX-1 showed direct lytic activity on human erythrocytes and platelets, whereas the succinylated or carbamylated derivatives did not show lytic activity. The loss of lytic activity cannot be related to conformational changes or phospholipid binding abilities of the modified cardiotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy

The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and [2-13C]-and [3-13C]phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy. Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed. The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and [2-13C]- or [3-13C]PEP has been examined by 13C NMR spectroscopy. With [2-13C]PEP, in addition to the resonances due to [2-13C]PEP and [8-13C]EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm. The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP. The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson [Levin, J. G., & Sprinson, D. B. (1964) J. Biol. Chem. 239, 1142-1150] and recently independently observed by Anderson et al. [Anderson, K. S., Sikorski, J. A., Benesi, A. J., & Johnson, K. A. (1988) J. Am. Chem. Soc. 110, 6577-6579]. The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate. With [3-13C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm. The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP. The resonance at 26.2 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS)     

A note on the use of microwaves in an improved non-radioactive DNA hybridization procedure for the detection of bacteria in foodstuffs

Foodstuffs were artificially contaminated with Escherichia coli carrying plasmid pBR322, dot blotted onto nylon membranes and briefly subjected to microwaves in the presence of 1·5 mol/l NaC1/0·5 mol/l NaOH. Subsequent hybridization with a biotin-labelled probe specific for pBR322 enabled the detection of cell concentrations > 10⁴ cells/dot blot, equivalent to 2 × 10⁷ cells/g food tested. This shortened and simplified method was effective for all ten foods tested, generated low background levels and should be applicable to a wide range of bacteria.     

Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure

The predicted conformation and position of the central transforming region (residues 55–67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1–166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an -helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.     

The chemical modification of cysteine-69 of rat liver fatty acid-binding protein (FABP): a fluorescence approach to FABP structure and function

Summary Hepatic-FABP was labelled at cysteine-69 with the fluorescent environmentally sensitive reporter group AEDANS. The labelled protein had an emission maximum at 465 nm indicating that cysteine-69 was buried in a non-polar environment. The modified protein was still able to bind ligands such as oleic acid, oleoyl CoA and haem. The affinity of AEDANS-FABP for haem was unaltered as compared with the native protein indicating that cysteine-69 must be remote from the ligand binding site. The binding of oleic acid did not significantly perturb the fluorescence emission spectrum of the fluorescent reporter group suggesting that there are not large conformational changes in the region of cysteine-69 on fatty acid binding. The binding of stoichiometric amounts of oleoyl CoA was accompanied by a small fluorescence enhancement which suggests that fatty acyl CoAs may interact with other regions of the FABP molecule not involved in fatty acid binding.Abbreviations FABP Fatty Acid-Binding Protein - AEDANS 5-[2-(Acetyl)aminoEthyl]Aminonaphthalene-1-Sulphonic Acid - AAEDANS 5-[2-(Iodoacetyl-AminolEthyl] aminonaphthalene-1-Sulphonic Acid - DTNB 5,5Dithiobis-(2-Nitrobenzoic acid)     

The effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold

The purpose of this investigation was to determine the effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold (PWCFT). Ten adult males (mean age 23 years, SD 3) volunteered as subjects for this study. During the first laboratory visit the subjects performed a maximal bicycle ergometer test for the determination of maximum oxygen consumption (VO2max). Between 48 and 72 h later, the subjects pedaled to exhaustion at a power output which corresponded to a mean of 76% of VO2max (range, 72-80%) for the purpose of glycogen depletion. For the next 3 days, the subjects were fed a 10.5 MJ.day-1 low carbohydrate diet which consisted of 7.5% carbohydrates, 22.0% protein and 70.5% fat. The subjects then performed an incremental cycle ergometer test to the onset of fatigue or PWCFT, which was estimated from integrated electromyographic voltages of the vastus lateralis muscle. For the next 3 days the subjects were fed a 10.5 MJ high carbohydrate diet which consisted of 72.2% carbohydrates, 12.4% protein and 15.4% fats for the purpose of glycogen supercompensation. The subjects then performed a second PWCFT test. A paired t-test indicated that there was no significant (p greater than 0.05) difference between the means of the PWCFT values (depletion 246 W, SD 30; supercompensation 265 W, SD 28) and they were highly correlated at r = 0.884. The results of this investigation suggested that the methods commonly used to affect glycogen depletion or supercompensation had no effect on PWCFT.