Integrity of polypeptide chains of spectrin from human erythrocytes.

The suggestion that the high molecular weight erythrocyte membrane protein, spectrin, consists of subunits resistant to dissociation by both sodium dodecyl sulfate and 6 m guanidine hydrochloride has been reevaluated. By gel electrophoresis in dodecyl sulfate and thin-layer gel filtration in 6 m guanidine hydrochloride as well as in the much more powerful denaturant guanidine thiocyanate, and by sedimentation velocity in 6 m guanidine hydrochloride, the molecular weight emerges in the range 2–2.5 × 10⁵. Denaturation profiles as a function of guanidine hydrochloride concentration, observed by circular dichroism, reveal that the spectrin conformation is unusually labile, with a mid-point for the unfolding process at a denaturant concentration near 1 m. Complete acylation with succinic anhydride, as well as reaction with citraconic anhydride, leaves the molecular weight unchanged even in 6 m guanidine hydrochloride. The possibility of measuring molecular weights of proteins by viscosity determination in trifluoroacetic acid was explored. A calibration with a series of proteins gave a Mark-Houwink plot with high scatter, which did not result from low precision of viscosity determination or protein degradation. Evidence is adduced from infrared spectra that the scatter is due to a variable degree of protonation of the polypeptide backbone in the acid, leading to altered hydrodynamic characteristics. Within the semiquantitive limits of the method, spectrin is not further disaggregated in trifluoroacetic acid. The presence of refractory noncovalent interactions and of covalent cross-links has been variously invoked to explain an apparent microheterogeneity in spectrin preparations. The results here described appear to render the former explanation untenable.     

Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low density lipoprotein (apoprotein B-PIII)

Human low-density lipoproteins (LDL) were isolated from single donors by differential centrifugation between densities of 1.020 and 1.050 g/mL. The LDL were reduced and alkylated in 7 M guanidine hydrochloride, and the lipid was removed by multiple extractions in the cold with a mixture of diethyl ether and ethanol. Sedimentation studies on the resultant human apoprotein B (apoprotein B-PI) at low concentrations in 6.00 M guanidine hydrochloride showed a single sharp boundary with a sedimentation coefficient of 2.15 +/- 0.04 S at 25 degrees C, uncorrected for viscosity or density. Diffusion experiments performed in the same solvent at low speeds in the analytical ultracentrifuge gave a D25 = 0.694 +/- 0.043 Fick. Combining these values with an apparent specific volume of 0.703 mL/g yielded a molecular weight of 387 000, indistinguishable from that obtained by sedimentation equilibrium analysis in 7 M guanidine hydrochloride. Similar values were also obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rat very low density lipoproteins (VLDL), isolated from sera of Triton WR1339 treated animals, were used as the source of rat apoprotein B-PIII. The delipidated VLDL were solubilized in sodium dodecyl sulfate, and apoprotein B-PIII was isolated by Sepharose 4B chromatography. With appropriate corrections for density and viscosity, the behavior of rat apoprotein B-PIII was identical, upon analytical ultracentrifugation, in 6 and 7.7 M guanidine hydrochloride, corresponding to sedimentation and diffusion coefficients of 1.47 S and 0.92 Fick, respectively, in 6 M guanidine hydrochloride. These data may be combined to yield a molecular weight of 210 000. Similar values were obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.     

Mechanism of reversible glycine cleavage reaction in Arthrobacter globiformis. Function of lipoic acid in the cleavage and synthesis of blycine.

The physical and chemical characterization of horse serum butyrylcholinesterase has been extended. The results show that the enzyme is a glycoprotein containing about 20% carbohydrate by weight. Mannose, glucosamine, galactose, and sialic acid are the sugar residues found. The extinction coefficient of butyrylcholinesterase, E_1cm^1% at 280 nm, was found to be 15.2 ± 0.3 by dry weight determination. The molecular weight of the protein in dilute phosphate buffer was determined to be (31.7 ± 1.2) × 10⁴ by high speed equilibrium sedimentation with a redetermined partial specific volume of 0.723 ± 0.003 ml/g. Subunit molecular weights for the dissociated protein were found to be (7.9 ± 0.4) × 10⁴ and (8.1 ± 0.1) × 10⁴, respectively, in guanidine hydrochloride and in a solution at pH 11.8. The subunit molecular weight was also estimated to be (8.8 ± 0.2) × 10⁴ by analytical sodium dodecyl sulfate-gel electrophoresis. This apparently higher subunit molecular weight from dodecyl sulfate gels is expected for glycoproteins containing significant amounts of carbohydrate. No free sulfhydryl group was detected, even though there are six half-cystines in each subunit. Therefore, it seems likely that there are three pairs of disulfide bonds per subunit. The available data indicate that native butyrylcholinesterase is a tetrameric glycoprotein consisting of subunits of equal molecular weight.     

Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

Quaternary structure of delta-aminolevulinic acid synthase from Rhodopseudomonas spheroides.

Delta-Aminolevulinic acid synthase (succinyl-CoA: glycine C-succinyltransferase (decarboxylating) EC 2.3.1.37) was purified from Rhodopseudomonas spheroides. The purity of the enzyme preparation was established by its behavior in disc electrophoresis in the presence and absence of sodium dodecyl sulfate and by analytical ultracentrifugation. The molecular weight of the enzyme as determined by sedimentation equilibrium was found to be about 80,300, a value similar to those obtained by gel filtration, polyacrylamide gel electrophoresis, and sucrose gradient centrifugation. The molecular weight of the enzyme, denatured with either sodium dodecyl sulfate or guanidine hydrochloride, was found to be about 45,000 and 41,000, respectively. The dimeric structure was supported by sedimentation in sucrose gradients. Further evidence for the dimetic nature of the enzyme was obtained by gel electrophoresis of the enzyme treated with dimethylsuberimidate and sodium dodecyl sulfate.     

Molecular weight of the major acidic glycoprotein of horse erythrocyte membrane

We describe a method for determining the subunit molecular weight of membrane glycoproteins which aggregate strongly in aqueous solutions. The sedimentation equilibrium technique which we use is completely thermodynamic in nature and makes no assumptions about the nature of the sedimenting species. We report experiments on the major acidic glycoprotein of horse erythrocyte membranes in 5 m guanidine hydrochloride. The partial specific volume and parameters describing the preferential interaction of the protein with guanidine are measured using a differential density method involving sedimentation equilibrium measurement in H₂O and in D₂O. We find the effective partial specific volume (φ′) to be rather small (0.30–0.38) implying strong preferential binding of guanidine to the protein. We find a molecular weight o1f 113,000 ± 10,000 which is in substantial disagreement with other estimates of the subunit molecular weight of the sialo protein of erythrocyte membrane. We conclude that either the subunits are so tightly aggregated that 5 m guanidine hydrochloride fails to disaggregate them or that the Na dodecyl sulfate-gel electrophoresis method leads to a substantial underestimate of the subunit molecular weight. Circular dichroism measurements indicate the helix content of the protein to be similar in 5 m guanidine and in phosphate buffer.     

Dissociation and reconstitution of human ferroxidase II.

The ferroxidase II protein from human serum is large and structurally complex. It possesses protein-bound lipid and copper components which are essential for the maintenance of its catalytic activity. Treatment of ferroxidase II with 8 M urea, 6 M guanidine hydrochloride, or 6 M guanidine hydrochloride and alkylation does not result in the dissociation of the enzyme into subunits. However, treatment with sodium dodecyl sulfate results in the dissociation of ferroxidase II into two nonidentical subunits, designated S-I and S-II. S-I contains little phospholipid, cholesterol, or copper and has a molecular weight of 3.8-3.9 X 10(5). In contrast, S-II contains bound phospholipid, cholesterol, and copper and has a molecular weight of 2.2-2.4 X 10(5). The lipid compositon of S-II is identical with the native enzyme. Sodium dodecyl sulfate-free S-I exhibits no ferroxidase activity. Immediately following removal of sodium dodecyl sulfate, S-II exhibits ferroxidase activity but S-II rapidly loses its activity in the absence of S-I. The separated subunits spontaneously reassociate upon removal of the sodium dodecyl sulfate to yield a fully active enzyme which chemically appears identical with native ferroxidase II. Furthermore, the reconstituted enzyme is stable. Both native and reconstituted ferroxidase II may be stored at 4 degrees C for 6 weeks without any loss in activity. This suggests that S-II, the copper and lipid-containing subunit, is the catalytic subunit and that S-I is essential for the stabilization of the enzymic activity of S-II. These results provide insight into the molecular structure and chemical composition of ferroxidase II and suggest that the complete native structure of ferroxidase II is required for the maintenance of i-s functional integrity.     

Association-dissociation of glycinin in urea,guanidine hydrochloride and sodium dodecyl sulphate solutions

The effect of urea, guanidine hydrochloride and sodium dodecyl sulphate on glycinin, the high molecular weight protein fraction from soybean has been investigated by analytical ultracentrifugation. Urea and guanidine hydrochloride dissociate the protein to a ‘2S’ protein through the intermediary 7S and 4S proteins. Howeαer, in sodium dodecyl sulphate the protein directly dissociates to a 2S protein. Analysis of the data by calculation of per cent fraction and S_20,w value indicates that dissociation and denaturation of glycinin occur simultaneously in the presence of the aboαe reagents but to different extents.     

The molecular weight of pig liver microsomal carboxylesterase

The enzyme carboxylesterase, isolated from the microsomes of pig liver, was found to have a molecular weight of 180,000 in dilute salt solutions as determined by the method of sedimentation equilibrium. In the presence of 6 m guanidine hydrochloride, 0.1 M β-mercaptoethanol, the molecular weight, uncorrected for preferential solvation, was found to be 61,000, also by the method of sedimentation equilibrium. The molecular weight determined for the enzyme (reduced and alkylated with acrylonitrile) in 6 m guanidine hydrochloride by the method of analytical gel chromatography was found to be 58,200. The method of disc gel electrophoresis in sodium dodecyl sulfate yielded a molecular weight of 62,000. The conclusion of the study is that the native carboxylesterase molecule is comprised of three subunits each with a molecular weight of approximately 60,000.     

Studies on the posterior silk gland of the silkworm Bombix mori. IV. Ultracentrifugal analyses of native silk proteins, especially fibroin extracted from the middle silk gland of the mature silkworm

Ultracentrifugal analyses of the native silk proteins extracted from the various parts of the middle silk gland of the mature silkworm have revealed that there exist four components with S°_20,w values of 10S, 9–10S, 9S, and 4S in the extract. It is suggested that the fastest 10S component is the native fibroin synthesized in the posterior silk gland and transferred to the middle silk gland to be stored there, while the slower three components probably correspond to inner, middle, and outer sericins which were synthesized in the posterior, middle, and anterior portion of the middle silk gland, respectively. Native fibroin solution was prepared from the most posterior part of the middle silk gland. Ultracentrifugal analyses have shown that the solution contains considerable amounts of aggregates in addition to the main 10S component. Treatment with lithium bromide (LiBr), urea, or guanidine hydrochloride solution up to 6 M all have failed to dissociate the 10S component. From the sedimentation equilibrium analyses and partial specific volume of 0.71₆, the molecular weight of the 10S component of the native fibroin solution was found to be between 3.2 – 4.2 x 10⁵, with a tendency to lie fairly close to 3.7 x 10⁵.     

Denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions

The denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions was examined by monitoring changes in the intrinsic fluorescence of tryptophan and tyrosyl residues. Changes in various fluorescence parameters, such as quantum yield, emission maximum, spectral half-width, fluorescence depolarization, and fluorescence quenching by acrylamide, have indicated that while phaseolin is relatively stable up to 8 M urea, it is completely destabilized in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate. Furthermore, while the denaturation of phaseolin in urea solutions followed a two-step process, that in guanidine hydrochloride and sodium dodecyl sulfate followed a single-step process. While the accessibility of tryptophan residues to the nonionic acrylamide quencher is almost 100% in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate, only about 72% was accessible in 8 M urea compared to 52% in native phaseolin. The results presented here suggest that the protomeric structure of phaseolin is quite stable to changes in the environment. This structural stability may be partly responsible for its resistance to proteolysis by various proteinases.     

Size distribution of poly(A)-containing messencer RNA from free polysomes of HeLa cells

The sedimentation properties of pulse-labeled and long-term labeled mRNA from highly purified HeLa cell free-polysomes, selected for poly(A) content by two successive passages through poly(T)-cellulose columns, were analyzed under native and denatured conditions. The sedimentation profile of the mRNA on both sodium dodecyl SO₄-sucrose gradients and formaldehyde-sucrose gradients showed a broad distribution of components with estimated molecular weights ranging from 2 × 10⁵ to 5.5 × 10⁶ daltons and a weight-average molecular weight of 8.5 × 10⁵ daltons.     

Evidence for an intermediate in the denaturation and assembly of phosphoglucose isomerase

The denaturation of dimeric rabbit muscle phosphoglucose isomerase in guanidine hydrochloride occurs in two discrete steps consisting of partial unfolding followed by subunit dissociation. In 3.5 to 4.5 m guanidine hydrochloride the enzyme forms a stable denaturation intermediate. Formation of this intermediate abolishes catalytic activity, shifts the protein fluorescence emission maximum from 332 to 345 nm, exposes all of the unavailable sulfhydryl groups, and decreases the s_20,w from 6.8 to 4.6 S. The intermediate dissociates into fully unfolded polypeptide chains with further increases in the concentration of the denaturant. The fluorescence maximum shifts to 352 nm and the s_20,w of the denatured monomer is 1.6 S. From the equilibrium constant for subunit association, 3 × 10⁴M^?1, in 4.7 m guanidine hydrochloride, the apparent free energy of association is estimated to be ?6 kcal mol^?1. Reconstitution of the enzyme protein takes place by the reversal of the steps observed upon denaturation. The denatured monomers refold and associate to reform the dimeric intermediate which then anneals to yield the intact enzyme molecule.     

Purification, characterization and reassembly of the bacteriophage T4D tail sheath protein P18.

P18, the sole component of T4 tail sheath, has been isolated in a monomeric active form from extended sheaths of intact tails which were dissociated at low ionic strength. The molecular weight of P18 is determined to be 65,000 from sedimentation equilibrium and 73,000 from sodium dodecyl sulphate/gel electrophoresis. Combining the diffusion constant (D_20,w = 5·5× 10^?7cm²s^?1)and the sedimentation constant (s⁰_20,w = 4·2 S) a value of 67,000 is obtained. The circular dichroism spectra reveal a striking similarity of the structure of P18 in the monomeric state and in the extended sheath conformation.The purified P18 is found to reassemble into extended sheaths if the core-baseplate complex is present, forming normal length tails. Structures similar to polysheath are formed in the absence of core-baseplates.     

The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg²⁺ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its K_ms for HPr and ATP are 31 μM and 1 mM, respectively.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Phytochrome: A Re-examination of the Quaternary Structure

Highly purified phytochrome samples from rye (Secale Cereale cv. Cougar) were fractionated by ultracentrifugation in isokinetic sucrose density gradients. Three protein species were separated with estimated sedimentation coefficients of 6.5S, 8.0S, and 11.5S. The 6.5S and 8.0S forms contained photoreversible phytochrome and produced a single subunit of 120,000 molecular weight upon reduction and electrophoresis in the presence of sodium dodecyl sulfate. The 11.5S species contained no detectable phytochrome. Reduction and electrophoresis of the 11.5S species in the presence of sodium dodecyl sulfate produced a major polypeptide of 32,000 molecular weight and a minor polypeptide of 48,000 molecular weight. The square tetrameric structures, observed by electron microscopy and previously thought to be phytochrome molecules, were found to be due to the presence of this 11.5S species in phytochrome preparations.     

Human kidney alpha-L-iduronidase: purification and characterization.

α-l-Iduronidase has been purified 25,000-fold from the soluble proteins of human kidney by chromatography on heparin-Sepharose, hydroxylapatite, and Bio-Gel P-100. The α-l-iduronidase activity is associated with 80% of the protein in the most purified preparation. It has a molecular weight of 60,000 ± 6500, determined by sedimentation equilibrium, and can be dissociated by reduction into subunits of molecular weight 31,000 ± 6500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate in the presence of dithiothreitol. It contains glucosamine and binds to concanavalin A. The pH optimum, K_m and V_max for two substrates, phenyl iduronide and [³H]anhydromannitol iduronide, were found to be 4.0, 1.05 mm, 16 μmol/mg protein/min, and 4·5, 9 mm and 270 μmol/mg protein/min, respectively. The enzyme is of the low uptake, noncorrective form with respect to fibroblasts cultured from the skin of patients with Hurler syndrome. It is inhibited by 10⁶ m p-chloromercuribenzoate and 10^?3 m Cu²⁺, but is not significantly affected by other divalent cations, EDTA, or sulfhydryl compounds. Antibodies to α-l-iduronidase have been raised in goats.     

Rat muscle 5'-adenylic acid aminohydrolase. I. Purification and subunit structure.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) has been purified to apparent homogeneity from rat muscle. The preparation exhibits a single polypeptide band with a molecular weight of 60,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a sedimentation coefficient of 11.3 S. Analysis by sedimentation equilibrium techniques showed the nat-ive enzyme to have a molecular weight of 238,000, whereas the enzyme, when analyzed in 6 M guanidine hydrochloride and 10 mM 2-mercaptoethanol, had a molecular weight of only 59,500. The amino acid composition of the enzyme was determined and peptide mapping was performed on a tryptic digest of S-carboxymethylated enzyme. NH2-terminal analysis by both the dansylation and cyanate procedures failed to identify a free NH2 terminus. Treatment of the enzyme with carboxypeptidase A resulted in the release of approximately 0.5 mol each of valine and leucine per 60,000 g of enzyme. The data presented indicate that hte native enzyme has a tetrameric structure consisting of four polypeptide chains each having a molecular weight of 60,000. The COOH-terminal analysis can be interpreted either as an indication of subunit heterogeneity or as a result of incomplete digestion of a -X-Leu-Val sequence at the end of a single type of polypeptide chain. Tryptic peptide maps strongly support the latter interpretation and suggest that the subunits are essentially identical.     

Molecular weight distribution profile of cartilage proteoglycan in aqueous guanidinium hydrochloride before and after carboxyl group modification.

The molecular weight distribution profile of a proteoglycan preparation (s^o_20.w = 23.1 S), isolated from bovine nasal cartilage in the presence of protease inhibitors, was studied by equilibrium sedimentation in 4 m guanidinium hydrochloride. Apparent reduced molecular weights ranged from 0.8 to 2.2 × 10⁶ and their concentration dependence appeared to be compatible with the presence of a heterogeneous population of self-associating macromolecules. Carbodiimide-induced modification of about 20% of the total carboxyl groups of the complex resulted in a shift of the molecular weight distribution profile, the new values ranging from 6 to 9 × 10⁵, with a marked predominance of the larger species. Exposure of the proteoglycan to carbodiimide or methylamine alone produced only a small shift of the apparent molecular weights. Moreover, chondroitin 4-sulfate molecules subjected to the same carbodiimide-promoted modification showed no significant change in their average molecular weights. It is therefore considered that the changes observed after carboxyl group modification cannot be attributed to cleavage of either the protein or the carbohydrate moiety of the proteoglycan complex. Rather, the evidence suggests that these functions are essential to the stabilization of the oligomeric species, which constitute a large proportion of the preparation even in 4 m guanidinium hydrochloride.