Improved expression,purification and characterization of VPR,a cold active subtilisin-like serine proteinase and the effects of calcium on expression and stability

Cloning into a pET 11a vector, followed by high-level expression of the cold adapted subtilase, VPR, utilizing the rhamnose titratable T7 system of Lemo21, resulted in a dramatic increase of soluble protein compared to the older system used. Expression optimization clearly shows the importance of calcium in the medium after induction, both for stability of the proteinase and cell health. Characterization of the purified enzyme obtained in a redesigned purification protocol which removed apparent RNA contaminants, resulted in a significantly higher value for k_cat than previously reported. The new recombinant protein exhibited slightly lower stability against thermal denaturation and thermal inactivation. Our results also indicate that two of the calcium binding sites have apparent binding constants in the mM range. Binding of calcium to the weaker of those two sites only affects resistance of the enzyme against irreversible thermal inactivation. Differential scanning calorimetry revealed a non-two-state denaturation process, with indication of presence of intermediates caused by unfolding of calcium binding motifs.     

Apparent unmasking of [3H]GDP binding in rat brown-fat mitochondria is due to mitochondrial swelling

The presence of and biochemical background for the so-called 'unmasking' phenomenon in rat brown-fat mitochondria was investigated (i.e. the apparent increase in [3H]GDP binding to the 'uncoupling' protein thermogenin, without a concomitant increase in the amount of the protein). It was found that an unmasking could be observed both 1 h after norepinephrine injection and after 1 h cold stress, provided that the rats were preacclimated to 28 degrees C. The unmasking could be observed both when a filtration method and when a centrifugation method for determination of [3H]GDP-binding capacity were used; however, the absolute values were higher with the filtration method. Based on observations of slower cytochrome-c oxidase sedimentation during centrifugation, the possibility that the matrix volume of brown-fat mitochondria isolated from warm-acclimated animals was smaller than that of cold-stressed animals was investigated with 3H2O. The cold stress increased the matrix volume from being nearly non-existent to about 1 microliter/mg. A preswelling procedure in an ionic medium could similarly increase the matrix volume in mitochondria from warm-acclimated animals but was without significant effect in the already swollen mitochondria from cold-stressed animals or from animals adapted to a lower temperature. In mitochondria from warm-acclimated animals, the ionic preswelling procedure was fully able to increase the apparent amount of GDP binding to that observed in mitochondria from cold-stressed animals, but it was practically without effect on GDP binding in mitochondria from cold-stressed animals or from animals acclimated to a lower temperature. It is concluded that the apparent 'unmasking' phenomenon, observed when the tissue is less activated than in normal control situations, is not (as hitherto anticipated) due to a specific change in thermogenin as such, but is a reflection of a general mitochondrial phenomenon.     

The concentration of uncoupling protein correlates with Mg2+ activated mitochondrial binding of GDP

Rats were housed at 4 degrees C for periods of up to 26 days. As little as 2 h of cold exposure caused an increase in the binding of [3H]GDP to mitochondria from brown adipose tissue. Incubation of mitochondria in vitro with 10 mM Mg2+ caused a marked increase in the subsequent binding of GDP to mitochondria from rats housed at 28 degrees C and a smaller increase in that from rats exposed to 4 degrees C for 2 h. Chronic exposure to cold led to an even greater increase in the amount of GDP bound to mitochondria incubated with Mg2+. The time course for the increase in the concentration of uncoupling protein was compared with that for GDP binding to mitochondria with and without Mg2+ treatment. The concentration of uncoupling protein appears to be correlated with the GDP-binding values for mitochondria treated with Mg2+ (r = 0.70) but not with the GDP binding to untreated mitochondria (r = 0.36). Therefore, the binding of GDP to untreated mitochondria may represent thermogenic activity at the time of death, whereas that after treatment with Mg2+ may more closely reflect total thermogenic capacity of the mitochondrion.     

Unmasking of GDP binding sites on hamster brown adipose tissue mitochondria and uncoupling protein.

1. A rapid unmasking of GDP binding sites on brown adipose tissue (BAT) mitochondria was observed when hamsters acclimatized to 28 degrees C were exposed to a temperature of 4 degrees C for 2 hr. 2. No rapid unmasking of GDP binding sites was observed when hamsters housed at 22 degrees C were briefly exposed to 4 degrees C. 3. The amount of GDP bound to BAT mitochondria from hamsters increased during 2 weeks of exposure to 4 degrees C, but did not change between 2 weeks and 30 days of cold exposure. 4. Incubation of mitochondria with 10 mM Mg2+ prior to the GDP binding assay increased the subsequent GDP binding to BAT mitochondria from hamsters housed at 28, 22 or 4 degrees C, albeit to different degrees. 5. The amount of GDP bound to uncoupling proteins isolated from untreated and Mg(2+)-treated mitochondria of hamsters and rats was measured. Scatchard analyses of the binding of GDP to purified uncoupling protein indicate that increases in the number of binding sites due to Mg2+ treatment of mitochondria do not change the affinity of the protein for GDP.     

The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein

A membrane-bound protein cofactor (ARF) is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory regulatory component (Gs) of adenylate cyclase. Improved methods for the purification of ARF from bovine brain are described. ARF has a high-affinity binding site for guanine nucleotides. Binding of GTP or GTP gamma S to ARF is necessary for the activity of the cofactor; GDP X ARF does not support ADP-ribosylation of Gs. Although the protein as purified contains stoichiometric amounts of GDP, GTPase activity of isolated ARF was not detected. Cholera toxin-dependent activation of adenylate cyclase thus requires two guanine nucleotide binding proteins.     

Uncoupling protein and its mRNA in brown adipose tissue of newborn rabbits

Guanosine diphosphate binding to the uncoupling protein of isolated mitochondria of brown adipose tissue in newborn rabbits was measured as an index of thermogenic activity. The binding was 0.281 +/- 0.022 nmol GDP/mg mitochondrial protein at 1 day of age, 0.214 +/- 0.017 at 3 days, 0.428 +/- 0.038 at 5 days, and 0.208 +/- 0.016 at 7 days. The increase in binding between 3 and 7 days of age suggests that the brown fat has an increased thermogenic capacity at that age. In addition, the potential for synthesis of the uncoupling protein was investigated in 1- to 5-day-old newborn rabbits by probing the total cellular ribonucleic acid for the messenger that codes for uncoupling protein. The amount of uncoupling protein messenger was highest at 1 day of age and declined at least until 5 days of age. Because the amount of uncoupling protein messenger decreased as the GDP binding increased, the results suggest that either the initially translated uncoupling protein was unmasked at about 5 days of age or there was a delay in the incorporation of uncoupling protein into the mitochondrial inner membrane, or both.     

Association of a GDP binding activity with initiation factor eIF-2 from calf liver

A highly purified preparation of the eucaryotic initiation factor eIF-2 from calf liver which forms a ternary complex with GTP and Met-tRNA_f^Met also exhibits a potent GDP binding activity. The factor preparation specifically forms a binary complex with GDP, other ribonucleoside diphosphates and GTP are inactive. Evidence is presented indicating that the GTP-dependent Met-tRNA_f^Met binding and binary complex formation with GDP are mediated by the same protein which has an apparent molecular weight of 67,000 as judged by glycerol density gradient centrifugation.     

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.     

Time-course variations of effective proton conductance and GDP binding in brown adipose tissue mitochondria of rats during prolonged cold exposure.

1. Time-course variations of the thermogenic pathway in rat brown adipose tissue (BAT) mitochondria were examined. 2. Several parameters of mitochondrial energization, protonmotive force and its components pH gradient and membrane potential were investigated. The specific binding of GDP was compared with the effective proton conductance (CmH+) of the membrane. 3. Ten-days cold exposure led to maximal GDP binding and GDP-dependent CmH+. 4. The subsequent relative decrease in GDP binding observed during prolonged cold exposure (40 days) was functional and led to a lower GDP-dependent CmH+. CmH+ showed greater variation than GDP binding. 5. The CmH+ decrease was not due to a masking of active sites of the uncoupling protein. 6. Basal GDP-independent CmH+ was not modified. 7. Results are discussed with reference to the significance of biochemical measures and to the physiological regulation of BAT thermogenesis.     

Differential interaction of fatty acids and fatty acyl CoA esters with the purified/reconstituted brown adipose tissue mitochondrial uncoupling protein

Proteoliposomes containing highly purified uncoupling protein generated by a modified purification/reconstitution procedure carried out active GDP dependent proton conductance. It was further established that long chain acyl CoA esters as well as fatty acids stimulated proton influx by the uncoupling protein, and, moreover, that the acyl CoA esters were partially effective in overcoming the inhibition by GDP. GDP binding to the purified uncoupling protein was inhibited by acyl CoA esters but not fatty acids. Phenylglyoxal which prevents GDP binding to the uncoupling protein eliminated the acyl CoA but not the fatty acid effect on proton conductance. These results substantiate the fact that nucleotides and acyl CoA esters act at the same regulatory site on the uncoupling protein, whereas, fatty acids act at a separate site. The properties of the purified/reconstituted uncoupling protein confirm they are identical to those inherent in brown adipose tissue mitochondria.     

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: application to Bruton's tyrosine kinase

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG‐binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin‐binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three‐tag system comprised of CBP, streptavidin‐binding peptide (SBP) and hexa‐histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP‐His₆ purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems.     

Cloning, Overexpression and Purification of Bacillus subtilis Elongation Factor Tu in Escherichia coli

To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His₆ tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.     

Nucleotide and metal interactions affecting inactivation of spinach chloroplast coupling factor by NaCl in the cold

The ability of isolated chloroplast coupling factor (CF₁) to hydrolyze ATP is destroyed when the enzyme is incubated in the cold. This inactivation is prevented completely by ATP at low concentrations and partially by adenosine imidodiphosphate. Other nucleotides, including ADP, GTP, GDP, and UTP, and also inorganic pyrophosphate, will prevent cold inactivation if certain divalent metal cations (Mg, Mn, Co, or Zn) are also present. These effects are taken as indications of binding of nucleotides to isolated CF₁ and the pattern of specificity is compared with those found in other experimental situations.     

Involvement of histidine residues in the substrate binding of elongation factor Tu from Thermus thermophilus: Proton nuclear magnetic resonance and photooxidation study

Proton nuclear magnetic resonance (nmr) spectra (270 MHz) were measured of polypeptide chain elongation factor Tu (EF-Tu) from an extreme thermophile, Thermus thermophilus. This protein was stable enough for a series of nmr measurements at temperature as high as 50 °C. For histidine C₂ protons, pH dependences of nmr chemical shifts were measured in the pH range from 5.5 to 8.0. The nmr titration curve of one histidine residue of free EF-Tu was markedly affected by the binding with GDP. This titration curve was further affected by the ligand substitution from GDP to GTP, indicating that this histidine is involved in the binding of EF-Tu with guanine nucleotides. The nmr titration curve of another histidine was also affected by the ligand substitution from GDP to GTP. The results of photooxidation experiments suggest that histidine residues are involved in the binding of EF-Tu with guanine nucleotides as well as with aminoacyl-tRNA and/or ribosomes.     

Human SLC26A4/Pendrin STAS domain is a nucleotide-binding protein: Refolding and characterization for structural studies

Mutations in the human SLC26A4/Pendrin polypeptide (hPDS) cause Pendred Syndrome /DFNB4, syndromic deafness with enlargement of the vestibular aqueduct and low-penetrance goiter. Here we present data on cloning, protein overexpression and purification, refolding, and biophysical characterization of the recombinant hPDS STAS domain lacking its intrinsic variable sequence (STAS-ΔIVS). We report a reproducible protein refolding protocol enabling milligram scale expression and purification of uniformly ¹⁵N- and ¹³C^/15N-enriched hPDS STAS-ΔIVS domain suitable for structural characterization by solution NMR. Circular dichroism, one-dimensional ¹H, two-dimensional ¹H–¹⁵N HSQC, and ¹H–¹³C HSQC NMR spectra confirmed the well-folded state of purified hPDS STAS-ΔIVS in solution. Heteronuclear NMR chemical shift perturbation of select STAS-ΔIVS residues by GDP was observed at fast-to-intermediate NMR time scales. Intrinsic tryptophan fluorescence quench experiments demonstrated GDP binding to hPDS STAS-ΔIVS with K_d of 178 μM. These results are useful for structure/function characterization of hPDS STAS, the cytoplasmic subdomain of the congenital deafness protein, pendrin, as well as for studies of other mammalian STAS domains.     

Purification and characterization of neurotensin receptor from rat brain with special reference to comparison between newborn and adult age rats.

Scatchard analysis of saturation curves was performed to compared newborn and adult rat neurotensin receptor using [3H] neurotensin as a tracer. The membrane fraction of newborn rat cerebral cortex has a single population of neurotensin receptor (Kd = 0.13 nM, Bmax = 710 fmol/mg protein), whereas adults have two distinct neurotensin binding sites (high affinity site, Kd1 = 0.13 nM; low affinity site, Kd2 = 20 nM). High affinity neurotensin receptor, solubilized with digitonin, was purified from newborn rat cortex by affinity chromatography. An overall purification of 14,000-fold was achieved. The binding of [3H] neurotensin to the purified receptor is saturable and specific, with a Kd of 0.45 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol revealed purified material of a single major band of Mr = 55,000.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Differences in thermal properties of NAD malate dehydrogenase in genotypes of Lathyrus japonicus Willd (Leguminosae) from maritime and continental sites

Abstract The response of the kinetic properties of NAD malate dehydrogenase (MDH) was compared for two clones of Lathy rus japonicus Willd. collected in two contrasting climatic sites in Eastern North America. MDH from the cold adapted maritime genotype (Hudson Bay, Québec) had lower thermostability, reduced apparent (E_a) and free (ΔG^?) energy of activation and lower specific activity when compared to MDH of plants from the warm summer continental site (Lake Michigan). Electrophoretic analyses show little differentiation in the isozyme profiles of the two genotypes. Thermostability differences were primarily associated with the mitochondrial isozymes which, however, were not differentiated electrophoretically Substrate binding ability of MDH, as measured by apparent Km, was more sensitive to high assay temperature in the cold adapted maritime genotype.     

A commentary on the interpretation of in vitro biochemical measures of brown adipose tissue thermogenesis

In this article we comment on the various in vitro biochemical measurements employed to assess the thermogenic activity and capacity of brown adipose tissue. The meaning and significance of changes in tissue weight, protein content, cell number, and mitochondrial mass are each summarized. In addition, various indices of the proton conductance pathway-mitochondrial swelling, proton conductance, uncoupling protein concentration, and GDP binding studies--are discussed. The issue of unmasking and masking of GDP binding sites is reviewed; recent reports have clearly demonstrated unmasking and masking, and it is concluded that GDP binding studies are an index of the activity of uncoupling protein, rather than a measure of its concentration. It is suggested that tissue mass, mitochondrial content, mitochondrial GDP binding, and uncoupling protein concentration represent core measurements for the biochemical assessment of the thermogenic activity and capacity of brown adipose tissue. Auxiliary measurements include Scatchard analysis of GDP binding data to distinguish changes in the number of binding sites from potential changes in Kd, and mitochondrial swelling studies, as an additional index of proton permeability. The distinction between thermogenic activity (GDP binding, proton permeability) and capacity (uncoupling protein content), both on a per unit of mitochondrial protein and per tissue basis, is emphasized.     

Activator proteins for sulphatide hydrolysis and GM1-ganglioside hydrolysis. Probable identity on the basis of their co-purification,properties, ligand binding and immunochemical interactions

The concurrent purification of the activator protein for sulphatide hydrolysis and for G_M1-ganglioside hydrolysis including chromat ofocusing and hydrophobic chromatography stages is described. The purified preparation has a pl of 4.2 and the sub-unit M_r is 10 000. The stoichiometry of binding of sulphatide and ganglioside to the protein is very similar. Both activities are removed in similar proportions on binding to IgG purified from antisera raised against the activator protein. The probable identity of the activator protein for sulphatide hydrolysis with that for G_M1-ganglioside hydrolysis and a molecular explanation for this identity are discussed.