Tris-Cl Buffer Research Articles

Purification and Initial Biochemical Characterization of ATP:Cob(I)alamin Adenosyltransferase (EutT) Enzyme of Salmonella enterica

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to approximately 70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (approximately 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 microm, kcat = 0.03 s(-1), and Vmax = 54.5 nm min(-1). Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 microm, kcat = 0.06 s(-1), and Vmax = 105 nm min(-1). Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was > or =50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the beta-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.

Fluorimetric determinations of nucleic acids using iron, osmium and samarium complexes of 4,7-diphenyl-1,10-phenanthroline

New sensitive, reliable and reproducible fluorimetric methods for determining microgram amounts of nucleic acids based on their reactions with Fe(II), Os(III) or Sm(III) complexes of 4,7-diphenyl-1,10-phenanthroline are proposed. Two complementary single stranded synthetic DNA sequences based on calf thymus as well as their hybridized double stranded were used. Nucleic acids were found to react instantaneously at room temperature in Tris–Cl buffer pH 7, with the investigated complexes resulting in decreasing their fluorescence emission. Two fluorescence peaks around 388 and 567 nm were obtained for the three complexes using excitation λ max of 280 nm and were used for this investigation. Linear calibration graphs in the range 1–6 μg/ml were obtained. Detection limits of 0.35–0.98 μg/ml were obtained. Using the calibration graphs for the synthetic dsDNA, relative standard deviations of 2.0–5.0% were obtained for analyzing DNA in the extraction products from calf thymus and human blood. Corresponding Recovery% of 80–114 were obtained. Student's t-values at 95% confidence level showed insignificant difference between the real and measured values. Results obtained by these methods were compared with the ethidium bromide method using the F-test and satisfactory results were obtained. The association constants and number of binding sites of synthetic ssDNA and dsDNA with the three complexes were estimated using Rosenthanl graphic method. The interaction mechanism was discussed and an intercalation mechanism was suggested for the binding reaction between nucleic acids and the three complexes.

Computer-assisted Docking of Flavodoxin with the ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme Reveals Residues Critical for Protein-Protein Interactions but Not for Catalysis

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA.FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA.FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.

Dodecameric Structure of the Small Heat Shock Protein Acr1 from Mycobacterium tuberculosis

Small heat shock proteins are a ubiquitous and diverse family of stress proteins that have in common an alpha-crystallin domain. Mycobacterium tuberculosis has two small heat shock proteins, Acr1 (alpha-crystallin-related protein 1, or Hsp16.3/16-kDa antigen) and Acr2 (HrpA), both of which are highly expressed under different stress conditions. Small heat shock proteins form large oligomeric assemblies and are commonly polydisperse. Nanoelectrospray mass spectrometry showed that Acr2 formed a range of oligomers composed of dimers and tetramers, whereas Acr1 was a dodecamer. Electron microscopy of Acr2 showed a variety of particle sizes. Using three-dimensional analysis of negative stain electron microscope images, we have shown that Acr1 forms a tetrahedral assembly with 12 polypeptide chains. The atomic structure of a related alpha-crystallin domain dimer was docked into the density to build a molecular structure of the dodecameric Acr1 complex. Along with the differential regulation of these two proteins, the differences in their quaternary structures demonstrated here supports their distinct functional roles.

Crystal Structure of the tRNA 3′ Processing Endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.

Proteomics Reveal Cochlin Deposits Associated with Glaucomatous Trabecular Meshwork

The etiology of primary open angle glaucoma, a leading cause of age-related blindness, remains poorly defined, although elevated intraocular pressure (IOP) contributes to the disease progression. To better understand the mechanisms causing elevated IOP from aqueous humor circulation, we pursued proteomic analyses of trabecular meshwork (TM) from glaucoma and age-matched control donors. These analyses demonstrated that Cochlin, a protein associated with deafness disorder DFNA9, is present in glaucomatous but absent in normal TM. Cochlin was also detected in TM from the glaucomatous DBA/2J mouse preceding elevated IOP but found to be absent in three other mouse lines that do not develop elevated IOP. Histochemical analyses revealed co-deposits of Cochlin and mucopolysaccharide in human TM around Schlemm's canal, similar to that observed in the cochlea in DFNA9 deafness. Purified Cochlin was found to aggregate after sheer stress and to induce the aggregation of TM cells in vitro. Age-dependent in vivo increases in Cochlin were observed in glaucomatous TM, concomitant with a decrease in type II collagen, suggesting that Cochlin may disrupt the TM architecture and render components like collagen more susceptible to degradation and collapse. Overall, these observations suggest that Cochlin contributes to elevated IOP in primary open angle glaucoma through altered interactions within the TM extracellular matrix, resulting in cell aggregation, mucopolysaccharide deposition, and significant obstruction of the aqueous humor circulation.

Bacteriophage P22 Tail Accessory Factor GP26 Is a Long Triple-stranded Coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.

Synthesis, Photophysical Properties, and Biomolecular Labeling Studies of Luminescent Platinum(II)-Terpyridyl Alkynyl Complexes

Three luminescent alkynylplatinum(II) terpyridyl complexes, [Pt(tBu3tpy)(C⋮C−C6H4−X-4)](OTf) (X = NH2 1, NCS 2, NHCOCH2I 3), have been synthesized and characterized. Their electrochemical and photophysical properties have been studied, and the molecular structure of 2 has also been determined by X-ray crystallography. Complexes 2 and 3 exhibit strong luminescence in various media, the origin of which has been tentatively assigned as a dπ(Pt) → π*(tBu3tpy) 3MLCT excited state, with some mixing of a π(C⋮CR) → π*(tBu3tpy) 3LLCT character. Human serum albumin (HSA) has been labeled by the ready reaction of the isothiocyanate and iodoacetamide functional groups in 2 and 3, to afford the respective bioconjugates, 2-HSA and 3-HSA. Both bioconjugates in 50 mM Tris-Cl buffer (pH 7.4) are highly colored and exhibit luminescence in the visible region upon photoexcitation.

Involvement of a Nine-residue Loop of Streptokinase in the Generation of Macromolecular Substrate Specificity by the Activator Complex through Interaction with Substrate Kringle Domains

The selective deletion of a discrete surface-exposed epitope (residues 254-262; 250-loop) in the beta domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SK(del254-262)). A kinetic analysis of SK(del254-262) revealed that its low HPG activator activity arose from a 5-6-fold increase in K(m) for HPG as substrate, with little alteration in k(cat) rates. This increase in the K(m) for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SK(del254-262). The involvement of kringles was further indicated by a hypersusceptibility of the SK(del254-262).plasmin activator complex to epsilon-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK.plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SK(del254-262).HPG was reduced by about 3-fold in comparison with that of nSK.HPG . Overall, these observations identify the 250 loop in the beta domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK.HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process.

UNCEIN MAY BE A POTENTIAL TARGET FOR SULFUR MUSTARD ALKYLATION

Severe blister formation in theepidermal and dermal junction is one of the characteristic cutaneous lesions caused by bis-2-chloroethyl sulfide (sulfur mustard, HD), a bifunctional alkylating agent. Uncein is a newly discovered anchoring filament-associated antigen that has been shown to be undetectable in all forms of junctional epidermolysis bullosa, therefore suggesting its role in maintaining the integrity of the epidermal-dermal basement membrane zone. To test uncein as a potential target for HD alkylation in HD-induced vesication, uncein was biosynthetically labeled with 35 S-methionine and purified by immunoprecipitation with a 19-DEJ-1 monoclonal antibody from the medium of normal human keratinocyte (NHEK) cultures. The 3 5 S-labeled uncein was incubated with 50 muL of 10.0 mg/mL of HD in ethanol (ETOH) or ETOH, which served as the control in 50 mM Tris-Cl buffer (pH 7.4) for 2 h. In addition, 10.0 mg/mL of HD was incubated with uncein pretreated with 3 mM sodium thiosulfate, an HD scavenger. Unce...

Substrate specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates.

The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (Km and Vmax) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol.

RNase H Activity of Human Hepatitis B Virus Polymerase Expressed inEscherichia coli

Human Hepatitis B Virus (HBV) replication is accomplished by its own polymerase. The HBV RNase H domain of HBV polymerase has been expressed inEscherichia coliand purified by affinity column chromatography. The MBP-RNase H fusion protein (43 kDa MBP plus 17 kDa HBV RNase H domain) was proved to be RNase H byin vitroactivity assay, inhibitor studies, and mutagenesis. The HBV RNase H domain represented the optimal RNase H activity in the presence of either 8 mM MgCl2or 16 mM MnCl2. In Tris-Cl buffer, the optimum pH for MBP-RNase H fusion protein is between 7.7 and 8.2. The MBP-RNase H fusion protein required 40 mM monovalent cation for its enzyme activity, whereas it showed lower activity at a salt concentration of more than 100 mM. Ribonucleoside Vanadyl complex (RAV) and 2′-deoxyadenosine 5′-monophosphate (dAMP) inhibited the RNase H activity. Moreover, the mutation of highly conserved amino acids in the HBV RNase H domain diminished the RNase H activity. These results clearly suggest that the RNase H activity is separable from viral HBV polymerase enzymatic activities.

Biostatic activity of Coix lacryma seed extract on Toxoplasma gondii in macrophages.

Water extract of Coix lacryma seeds (Co-Ex) was separated into several components; dissolved with Tris-Cl buffer and the supernatant (WC1), ammonium sulfate treatment supernatant (WC2) and the pellet (WC3), QAE column chromatography of WC1 and the peak portions; WC4, WC5 and WC6. Murine peritoneal macrophages in DMEM containing 10% heat-inactivated FCS were infected with tachyzoites of Toxoplasma gondii, RH strain, in vitro. By adding modulators such as Co-Ex, WC1,2,3,4,5,6 and LPS or IFN-gamma for 24 hrs, toxoplasmastatic activity of macrophages was examined in relation to nitrite production. Nitrite production of macrophages was enhanced especially in the series of WC2, WC1 and the combination sample (WC1+WC2+WC3) by order, than other components or fractions (WC4, WC5, WC6) tested. Toxoplasmastatic actions such as percentage of the macrophages infected by T. gondii and fold increase of T. gondii in macrophages showed retroverse relations with the amount of nitrite production; i.e., as nitric oxide (NO) increased the phagocytic index of macrophages and the fold increase of tachyzoites in macrophages decreased. Nitrite (NO2) production was increased by adding IFN-gamma in all cases together with enhancement of biostatic effects. Through the results obtained, it is speculated that some components other than the non-proteinous and defatted components in Coix lacryma seeds may contribute to activate macrophages through induction of NO for the biostatic activity.

Essential sulfhydryl group of malic enzyme from Escherichia coli.

The activity of malic enzyme from Escherichia coli was unaffected by the monovalent cations Na+ or Li+ at 10 mM. At 100 mM, Li+ or Na+ inhibited the enzyme activity by 88% and 83%, respectively. However, the enzyme activity was stimulated by 40-80-fold with 10 mM K+, Rb+, Cs+, or NH4+. Less stimulation was observed with 100 mM of these stimulating cations. The stimulatory effect was lost after the enzyme was dialyzed against Tris-Cl buffer, but was regained after incubating the dialyzed enzyme with dithiothreitol. The regenerated enzyme was inactivated by 5,5'-dithiobis(2-nitrobenzoic acid). The resulting inactive thionitrobenzoyl enzyme could be regenerated to the active thiol-enzyme by dithiothreitol or converted to the inactive thiocyanoylated enzyme by KCN. The thiocyanoylated enzyme was insensitive to K+ stimulation, which suggested the essentiality of the sulfhydryl groups of the E. coli malic enzyme.

Divalent cation-sensitive antagonist binding to heart muscarinic receptors

1. 1. The binding of (−)[ 3H]quinuclidinylbenzilate (QNB), a potent muscarinic antagonist, to cardiac muscarinic receptors was examined in washed particles and microsomes isolated from rat heart atria. 2. 2. Addition of divalent cations (Mg 2+, Mn 2+ and Ca 2+) to the binding assays increased (6 to 8-fold) the antagonist binding to microsomes but exerted little or only weak (up to 35%) stimulation of the antagonist binding to washed particles. 3. 3. Mg 2+ ions present only in the dilution and washing buffer prior to rapid filtration of the binding reaction were capable of markedly increasing the antagonist binding to microsomes but not to washed particles. 4. 4. Such differential action of Mg 2+ on antagonist binding to subcellular fractions was seen when fractions were either isolated and/or incubated in media containing either 10 or 50 mM imidazole-Cl, Tris-phosphate or Tris-Cl buffer, each at pH 7.5. While the modulatory effect of Mg 2+ was not seen in Na-K-phosphate buffer, pH 7.5, the antagonist binding per se was much higher in Na-K-phosphate buffer compared to other buffers.

Distribution of β-endorphin-like and adrenocorticotropin-like activities in various tissues of the winter flounder, Pseudopleuronectes americanus

1. Different tissues of the winter flounder were extracted with an acidic medium (1 N acetic acid in 0.1 N HCl), 2. ACTH-like activity judged by the ability to stimulate corticosterone production by isolated rat adrenal cells, and opiate receptor binding activity, were present in extracts of the pituitary and brain. 3. Opiate receptor binding activity was absent from the gonadal extract but present in extracts of the heart, liver, interrenal and intestine. 4. β-Endorphin-like immunoreactivity was present in all of the afore-mentioned tissues. 5. Flounder pituitaries were also extracted with Tris-Cl buffer (pH 7.8) and the extract subjected to affinity chromatography on Con A-Sepharose to remove glycoproteins and ultrafiltered to remove high molecular weight materials. 6. When the resulting fraction (mol. wt < 10,000 but >2000) was gel filtered on Sephadex G-10, the unretarded peak contained ACTH-like bioactivity, opiate receptor binding activity and β-endorphin-like immunoreactivity.

Effects of sodium, lithium, and magnesium onIn vitro binding of [3H]SCH23390 in rat neostriatum and cerebral cortex

The effects of sodium, lithium, and magnesium on the in vitro binding properties of the D1 antagonist [3H]SCH23390 were examined with membrane preparations from rat neostriatum (CPU; caudate-putamen) and cerebral cortex (CTX). The saturation binding isotherms for both tissues performed in the presence of 120 mM of either Na+ or Li+ revealed an increase in the affinity, as compared to that observed when the incubation buffer was composed of Tris-Cl 50 mM with MgCl2 1 mM alone. For the CPU there were no changes in the maximum binding capacity (Bmax) in the different buffers used. In the case of the CTX, there was a loss of [3H]SCH23390 binding sites when either Na+ or Li+ 120 mM were added to the incubations, suggesting a lack of selectivity of this ligand in the absence of group IA cations. The agonist state of the [3H]SCH23390 binding site was studied in competition experiments with dopamine. The highest agonist affinity was obtained in 50 mM Tris-Cl buffer with 1 mM MgCl2 while the addition of 120 mM of either Na+ or Li+ caused a 3- to 5-fold decrease in the potency of dopamine to compete with specific [3H]SCH23390 binding in both CPU and CTX. The presence of magnesium was essential for the competition experiments; i.e.: a concentration of 1 mM MgCl2 was optimum to obtain dopamine antagonism of ligand binding, while increasing Mg2+ to 2 or 5 mM did not appear to further improve the inhibitions. The results support both agonist and antagonist affinity shifts for the dopamine D1 receptor labeled with [3H]SCH23390.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat liver ATP-sulfurylase: Purification, kinetic characterization, and interaction with arsenate, selenate, phosphate, and other inorganic oxyanions

ATP-sulfurylase (ATP:sulfate adenylyltransferase; EC 2.7.7.4), the first enzyme of the two-step sulfate activation sequence, was purified extensively from rat liver cytosol. The enzyme has a native molecular mass of 122 ± 12 kDa and appears to be composed of identical 62 ± 6-kDa subunits. At 30 °C and pH 8.0 (50 m m Tris-Cl buffer containing 5 m m excess Mg 2+), the best preparations have “forward reaction” specific activities of about 20 and 2 units × mg protein −1 with MoO 4 2− and SO 4 2−, respectively. The reverse (ATP synthesis) specific activity is about the same as the forward molybdolysis activity. The kinetic constants under the above conditions are as follows: KmA = 0.21 mM, K ia = 0.87 mM, K mB = 0.18 mM, K mQ = 0.65 μM, K iq = 0.11 μM, and K mP = 5.0 μM where A = MgATP, B = SO 4 2−, Q = APS, and P = total PP iat 5 mM Mg 2+. PP i is a mixed-type inhibitor with respect to MgATP and SO 4 2−. SeO 4 2− is an alternative inorganic substrate with a V max about 20% that of SO 4 2−. The product, APSe, is unstable. But in the presence of a sufficient excess of APS kinase, APSe is completely converted to PAPSe. The rate constant for nonenzymatic PAPSe hydrolysis was determined from measurements of the final steady-state reaction rate in the presence of limiting initial SeO 4 2− and a large excess of MgATP, ATP sulfurylase, APS kinase, and the other coupling enzymes and their cosubstrates. The results yielded a k of 2.4 ± 0.5 × 10 −3 sec −1 ( t 1 2 ca. 5 min). Phosphate is an effective buffer for enzyme purification and storage but inhibits catalytic activity, particularly at low substrate concentrations. In the presence of buffer levels of P i, the MgATP reciprocal plot of the SO 4 2−2-dependent reaction is concave-up. Inorganic monovalent oxyanions are dead end inhibitors competitive with SO 4 2−2 and apparently uncompetitive with respect to MgATP. The relative potencies are in the order ClO 3 2− > ClO 4 − > FSO 3 − > NO 3 −. Thiosulfate is also competitive with SO 4 2−2 but noncompetitive with respect to MgATP. Several divalent oxyanions (MoO 4 2−2, WO 4 2−2, CrO 4 2−2, and HAsO 4 2−2) promote the enzyme-catalyzed cleavage of MgATP to AMP and Mg PP i. The ratio V maxf K mA ranged from 0.7 to 200 for various reactive inorganic substrates. The cumulative results suggest the random binding of MgATP and the inorganic substrate but the ordered release of Mg PP rmi before APS. However, the relative contributions of the E → EA → EAB and E → EB → EAB paths cannot be evaluated. Chemical modification and inactivation protection studies suggest the presence of functionally essential argininyl, lysyl, and histidinyl residues.

Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity

The application of a simple, rapid, and inexpensive colorimetric growth assay was tested for human epidermal cells subcultured in uncoated plastic dishes. Cell layers were incubated with a crystal violet (CV) solution (0.2% with ethanol 2% in 0.5 M Tris-Cl buffer, pH 7.8) for 10 min at room temperature. After rinsing with 0.5 M Tris-Cl (pH 7.8) the cell layer was dried and decolorized with a sodium-dodecylsulfate solution (0.5% with ethanol 50% in 0.5 M Tris-Cl, pH 7.8) for 60 min at 37 degrees C. The extinction of the supernatant was read at the absorption maximum of 586 nm. The protein content of attached cells as classical parameter for quantifying cell growth was strongly related to CV extinction with a correlation coefficient of r = 0.98. Furthermore, the subcellular protein binding qualities of CV were analyzed. The water-soluble protein fraction of cultured epidermal cells was separated by sodium-dodecylsulfate polyacrylamide gel electrophoresis and stained with CV. We found a staining pattern which was qualitatively very similar to that of Coomassie blue, however less intense. Keratin electrophoresis revealed an affinity of CV to the 48, 50, and 56 kD cytokeratins. In conclusion, this CV assay is a reliable and simple method for the monitoring of epidermal cell growth in cultures.

Mitochondrial ATP synthase. Interaction of a synthetic 50-amino acid, beta-subunit peptide with ATP.

A 50-amino acid peptide predicted by chemical modification studies of F1 and by comparison with adenylate kinase to comprise part of an ATP-binding domain within the beta-subunit of mitochondrial ATP synthase has been synthesized and purified. In the numbering system used for bovine heart beta, the peptide consists of amino acid residues from aspartate 141 at the N-terminal end to threonine 190 at the carboxyl end. In Tris-Cl buffer, pH 7.4, the peptide undergoes a dramatic reaction with ATP resulting in precipitate formation. Analysis of the precipitate shows it to contain both peptide and ATP. Similar to the ATPase activity of F1 and the binding of nucleotide to the enzyme, the capacity of ATP to induce precipitation of the peptide is decreased markedly by lowering pH. Interaction of the peptide with the fluorescent ATP analog, TNP-ATP (2'(3')-O-(2,4-6-trinitrophenyl)-adenosine 5'-triphosphate), can be demonstrated in solution at low concentrations. A 7-fold enhancement in fluorescence is observed when 2.5 microM TNP-ATP interacts with 2.5 microM peptide. Divalent cation is neither required for ATP-induced precipitation of the peptide nor for demonstrating interaction between TNP-ATP and peptide, just as Mg2+ is not required for nucleotide binding to F1. These results indicate that the beta-subunit peptide studied here comprises at least part of a nucleotide-binding domain within the mitochondrial ATP synthase complex.