Isolation and preliminary characterization of electrophoretic variants of copper,zinc superoxide dismutase

Summary Three electrophoretic variants of superoxide dismutase can be detected in bovine erythrocytes by gel electrophoresis and electrofocusing. The two major forms, having isoelectric points at pH 5.2 and 4.9, were isolated by preparative focusing or chromatography. No differences were found in molecular weight, metal content, antigenicity, electron spin resonance spectrum, visible and ultraviolet optical spectra. In contrast, holo- and apo-superoxide dismutase, which have an electrophoretic mobility similar to that of the two major forms, showed unresolved isoelectric points but significantly different antigenicity. This result suggests that their different electrophoretic mobility is mainly conformation-related. The variant with pl 5.2, corresponding to the protein purified by ordinary procedures, was found to be inactivated by heat treatment faster than the other form. The latter one, on the other hand, gave rise to a multiple pattern of electrophoretic bands after incubation at 75 °C.It is suggested that superoxide dismutase multiplicity in erythrocytes is not genetically determined, but may be related to segregation of subunits, made non-identically by post translational asymmetrical modification.     

Electrophoretic Transfer from Polyacrylamide Gel to Nitrocellulose Sheets, a New Method To Characterize Multilocus Enzyme Genotypes of Klebsiella Strains

A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-μl samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of Klebsiella strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains of Klebsiella pneumoniae and 24 strains of Klebsiella oxytoca demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains.     

Detection of DNA-binding proteins in polyacrylamide gel after denaturing electrophoresis]

A simple method for detection of DNA-binding proteins is offered. These proteins can be revealed, following their electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gel containing labeled DNA, by washing the gel in buffer to remove SDS and to allow protein renaturation. Protein-free DNA is washed out, remaining in the DNA-binding proteins that restored their original characteristic. After autoradiography these proteins are seen as black bands (by one-dimensional gel electrophoresis) or spots (by two-dimensional gel electrophoresis) on a grey background. High sensitivity of the method is shown by using protein fractions of rat liver and a standard method.     

The usefulness of the analytical electrofocusing in a thin-layer polyacrylamide gel (PAG) in the histochemistry of enzymes cleaving peptide bonds

Summary The usefulness of the analytical electrofocusing in a thin-layer polyacrylamide (PAG) plate is shown on the basis of experiments with 10%–20% homogenates of various rat, rabbit and human organs as well as in lysates of isolated human lymphocytes and leucocytes in 2% Triton X100. 0.1–0.3 l of 12000 g supernatants were applied on LKB Ampholine PAG plates pH range 3.5–9.5 and subjected to electrofocusing. Afterwards portions of PAG plates were processed in optimized histochemical media for the demonstration of enzymes cleaving peptide bonds using various substrates. The same media were used in the histochemical detection of enzymes in sections on slides or semipermeable membranes. Electrofocused zymograms display species and organ differences. Ala-MNA, Leu-MNA and Met-MNA furnish similar zymograms. Bands obtained with Ala-MNA are most intense. Zymograms with Gly-Pro-MNA and Lys-Pro-MNA at pH 7.2 are not entirely identical. The majority of bands is more intense when Gly-Pro-MNA is used as the substrate and is due to the activity of DAP IV. The anodal band(s) focusing around pH 4.9 (rat) or 5.5 (man) is (are) much stronger with Lys-Pro-MNA and DAP II is responsible for it (them). Zymograms with His-Ser-MNA and Lys-Ser-MNA are similar. However, they differ form those revealed with Gly-Pro-MNA and Lys-Pro-MNA. Zymograms of lysates of leucocytes obtained with naphthol AS-D-chloroacetate and Ac-Ala-l-naphthyl ester are not identical showing that more than one enzyme is responsible for the bands. Results obtained with closely related substrates such as Ac-Ala-l-naphthyl ester and Ac-Met-l-naphtyl ester are not identical either. Zymograms of lysates of human lymphocytes revealed with Gly-Pro-MNA at pH 7.2 and Lys-Ala-MNA or Lys-Pro-MNA at pH 5.5 or 5.3 respectively show clearly the presence of DAP IV and DAP II in these cells. The analytical electrofocusing in PAG plates is a very useful tool in the histochemistry (and biochemistry) of enzymes cleaving peptide bonds. It helps very much in the evaluation of the substrate specificity, choosing of the discriminating substrate and enables a quick and reliable testing of the quality of various batches of commercially supplied substrates, diazonium salts and other reagents. The correlation of zymograms with the in situ pattern helps in the elucidation of the origin of individual bands in zymograms and suggests different molecular forms of peptidases in different localizations.     

Purification of intrinsic factor receptor from pig ileum using as affinity medium human intrinsic factor covalently bound to Sepharose

Intrinsic factor receptor was purified from hog ileum using human intrinsic factor covalently bound to Sepharose. A yield of 49.6% and a specific activity of about 2500 pmol/mg protein were achieved. The purified receptor was very unstable: 24 h of storage or addition of sodium phosphate precipitated it. The association constant of the receptor for the cyano[57Co]cobalamin-intrinsic factor complex was estimated to be 2.1 nM-1. In native polyacrylamide gel electrophoresis it resolved in two 256 and 320 kDa bands; beta-mercaptoethanol treatment cleared it into four bands corresponding to molecular masses of 107, 81.8, 63.5 and 53.2 kDa. An additional 39.3 kDa band was considered to be an artefact due to the presence of Triton X-114. Isoelectric focusing polyacrylamide gel electrophoresis resolved the receptor into two isoproteins isoelectric at pH 4.7 and 5.1. A similar result was obtained in column electrofocusing with the 125I-iodinated receptor. The 125I-labelled receptor did not crossreact with rabbit anti-human intrinsic factor antiserum. The electrophoretic properties of the receptor purified with intrinsic factor covalently bound to Sepharose were compared to those of the receptor purified by the use of the classical cobalamin-affinity medium. It was concluded that a disassembled receptor was produced using the classical method.     

One-lane sequence analysis of oligodeoxyribonucleotides

Treatment of 5'-end 32P-labeled oligodeoxyribonucleotides with 0.4 M aqueous piperidinium formate, pH 2, at 37 degrees C for 6 h, followed by treatment with 1 M aqueous piperidine at 90 degrees C for 6 h, produces, after electrophoresis through 27% polyacrylamide sequencing gels, one-dimensional distributions of radioactivity from which the base sequences can be deduced. The order of intensities for the bands signaling the various bases is G greater than A greater than C greater than T. The spacing from a given band to the next higher band in the ladder was base characteristic, the order of band spacings being G greater than T greater than or equal to A greater than C. In contrast to the one-cleavage one-lane DNA sequencing method reported earlier (B. J. B. Ambrose and R. C. Pless, 1985, Biochemistry 24, 6194-6200), which was based on treatment of end-labeled DNA with hot aqueous piperidine in the presence of sodium chloride, the present method produces a salt-free hydrolysate, thus minimizing electrophoretic irregularities in the fastest moving bands.     

Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis

We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein-containing bands or spots are cut out and inserted directly into a gas-phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150-pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels.     

Resolving Power: A Quantitative Measure of Electrophoretic Resolution

Resolving power is a quantitative measure of the ability of an electrophoretic system to separate DNA (and other) molecules of similar size. It is a dimensionless quantity, and hence facilitates comparison of the performance of electrophoretic systems that operate very differently. Resolving power can be determined as a function of molecular length from experimental data consisting of a series of completely resolved bands on a gel or blot; closely spaced bands are not required. We discuss factors such as the mass of DNA in a particular band and the spatial resolution of the system used to image the distribution of DNA on a gel or blot that, while not an intrinsic part of the electrophoretic system, may influence the observed resolving power. We derive an empirical global dispersion function that applies both to images of gels obtained after a fixed time of electrophoresis of all the samples and to images obtained as each species reaches a detector located at a fixed distance from the starting well. We use this dispersion function to show that the improvement in resolving power produced by extending the time or distance of electrophoresis in a static, uniform electric field asymptotically approaches a limiting value that is a function of the length of the DNA. When plotted as a function of molecular length, this limiting value defines an envelope that characterizes the intrinsic limits of performance of a particular electrophoretic system (e.g., electric field strength, gel type and concentration, buffer, temperature). Comparing the resolving power of static field agarose gel electrophoresis as routinely practiced for separating DNA molecules from 10³ to 10⁵ bp long with other electrophoretic schemes suggests that significant improvements should be achievable.     

Purification and partial characterization of ubiquitin-activating enzyme from Saccharomyces cerevisiae

Ubiquitin-activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin-Sepharose followed by HPLC anion-exchange chromatography. Enzyme activity was monitored by the ubiquitin-dependent ATP: 32PPi exchange assay. The purified enzyme has a specific activity of 1.5 mumol 32PPi incorporated into ATP.min-1.mg-1 at 37 degrees C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al. (1982) J. Biol. Chem. 257, 2537-2542. The catalytic activity showed a maximum at pH 7.0. Its molecular weight both in non-denaturing and in SDS-gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form. The isoelectric point determined by gel electrofocusing was approximately 4.7. Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant. The same two bands were also found in anti-E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.     

Recovery of sodium dodecyl sulfate-proteins from gel electrophoretic bands in a single electroelution step for mass spectrometric analysis

Mass spectrometric analysis of proteins derived from bands in gel electrophoresis is incompatible with the covalent fluorescent labeling of the protein. Thus, if one wishes to take advantage of the capacity for computer-directed electroelution of electrophoresis apparatus with intermittent fluorescent scanning of the migration path, the protein must be labeled fluorescently in a noncovalent, reversible fashion. This was recently achieved by staining of SDS-proteins with Cascade blue and electrophoresis in barbital buffer. However, the method was not a practical one for the purpose of isolating proteins from gel electrophoretic bands and their transfer into the mass spectrometer for three reasons: (i) Ten consecutive electroelution steps were required to obviate pH changes in the electroelution chamber; (ii) electroeluates from six gel electrophoretic lanes needed to be pooled; (iii) excessive protein loads ranging from 7 to 33 microg/pool were required. The present study reports the solution to those three problems. Mass spectrometric (MALDI-TOF) characterization of five proteins was demonstrated (i) after a single electroelution step; (ii) using electroelution from a single gel of 0.3-cm(2) cross-sectional area; and (iii) using a protein load of 2 (in one case 4) microg. However, the migration rates of the Cascade blue-SDS-protein-barbital complexes derived from proteins with widely varying molecular weights proved to be the same. Thus, despite the three advances made, the method to date remains restricted to samples of single proteins.     

Specific dimerization of the light chains of human immunoglobulin

1. The light chains of human immunoglobulin were allowed to dimerize in vitro on removal of the dispersing agents acetic acid or urea. 2. On electrophoresis in polyacrylamide gel at pH8.8 the dimers yielded up to nine regularly spaced bands. This approximates to the number of electrophoretic components known to occur among the monomers. 3. Single electrophoretic components of the dimers were isolated from the gel, dissociated into monomers, and subjected as such to electrophoresis in urea-containing gels. Each gave two adjacent bands. 4. Similarly, after all the light chains as monomers had been subjected to electrophoresis in urea-containing gels, single electrophoretic components were isolated and allowed to dimerize. When examined now as dimers in the absence of urea, each component gave two adjacent bands. 5. These findings are explicable on the following basis. (a) The dimerization of the light chains is specific, at least inasmuch as it occurs between monomers of the same electrophoretic mobilities. (b) With the buffer constant, different light chains undergo different changes in net charge on being transferred from urea-containing to urea-free solution; in this way two different chains of the same initial charge can acquire a charge difference of 1. 6. Experiments with Bence-Jones proteins and other homogeneous light chains gave results substantiating the conclusions (a) and (b).     

Gel electrophoretic investigations of prolamins in eu- and alloplasmatic octoploid primary triticale forms

 Unreduced prolamin fractions of eu- and alloplasmatic octoploid triticale forms were investigated by means of gel electrophoresis. The electrophoretic separation of the prolamin fractions was carried out by using one-dimensional, horizontal, native acidic polyacrylamide gel electrophoresis (APAGE) and gradient gels. A comparison of the electropherogram patterns of triticale forms with those of the genome donors showed that most of the parent prolamins can be found again in the corresponding triticale. However, the bands of the triticale forms exhibited lower intensities than those of the genome donors. On average the gliadin bands of the triticale forms showed equal reductions in intensities, thereby preserving the relationships of the band intensities in most cases. On the other hand, secalin bands which came from the rye parent showed varying reductions in intensities. A comparative analysis of the band patterns revealed that differences could often be found between triticale forms with the same genomic constitution. Often it concerned differences in intensity. In some cases the bands showed changes in mobility or a splitting up into two sub-bands. Starting with the plant material presented here we developed a new nomenclature of the prolamin band patterns of the triticale forms. Received: 15 July 1997 / Accepted: 23 July 1997     

Electrophoretic separation of plasma lipoproteins in agarose gel

A method has been developed for the separation of serum or plasma lipoproteins by electrophoresis in an agarose-agar gel mixture. The gel is applied to the surface of a thin polyester photographic film strip. With minor alterations in technique either single samples on individual strips or many samples on one large sheet may be processed. After fixation and dehydration the transparent film is stained with Sudan Black B and washed with water. The finished electrophoretogram can be obtained in 5 hr and consists of widely separated bands of lipoprotein fractions on a colorless transparent background, ideally suited for scanning with a densitometer. Plasma samples from different subjects show pre-beta lipoproteins of different mobilities. An effect of gel concentration on the extent of lipoprotein migration is demonstrated. The clearcut separation of lipoproteins by this method will facilitate the classification of hyperlipoproteinemias and improve quantitative estimates of lipoprotein distribution.     

Protein metabolism of Drosophila melanogaster male accessory glands—I: Characterization of secretory proteins

The electrophoretic properties of male accessory gland proteins of Drosophila melanogaster were studied in both the native and the denatured state. The molecular weights and the isoelectric points were determined. In addition, the relative abundance of individual fractions was measured. More than 40 protein bands were observed on one-dimensional dissociative gels, approx. 85 proteins were separated on two-dimensional gels. Secretions and epithelia of both the accessory gland and the ductus ejaculatorius each contain a distinct complement of proteins. The majority of accessory secretion proteins are basic. In the native state they are only soluble with difficulty. In the presence of urea, however, 21 fractions can be separated by one-dimensional electrophoresis with a low-pH buffer system. For two-dimensional separation non-equilibrium pH gel electrophoresis gave best results. All but one of the proteins of the ductus ejaculatorius secretion are acidic. The epithelial proteins were found to be acidic.     

Comparative studies of pig platelet membrane proteins

The proteins and glycoproteins of pig platelet membranes have been studied using gel electrophoretic techniques. A nomenclature is suggested from the apparent molecular weights estimated by one-dimensional electrophoresis. Isoelectric focusing showed that the majority of the proteins are in the 4.0-7.0 pH range. Subunits have been inferred from oligoproteins by two-dimensional, reduced-nonreduced, electrophoresis techniques. High resolution two dimensional electrophoresis combining isoelectric focusing and sodium dodecyl sulphate allows the observations of 60 polypeptide bands. An identification of some of those bands based on a correlation from reported human blood platelet membrane proteins is presented for comparison.     

Microspectrophotometric and enzymatic analyses of fish plasma proteins electrophoretically separated in thin polyacrylamide gels

A system for horizontal, discontinuous electrophoresis in thin sheets of polyacrylamide gel was developed to permit rapid and direct comparison of multiple samples of fish plasmas for population studies. The resulting electropherograms are suitable either for screening for enzyme polymorphisms or for densitometric scanning after gels are stained for total proteins. Each gel sheet (110×90×0.8 mm) can be used for simultaneous separation of 10–15 different samples. Since total voltage and the running time required for successful resolution of fine protein bands are greatly reduced in this system, minimal heating occurs within the gel matrix during electrophoresis. Up to 120 different blood samples (8 gel sheets) can be processed in about 8 h. Gel sheets are routinely stained overnight in Coomassie Brilliant Blue (0.1%, v/v), rinsed several times and stored overnight in 7% acetic acid to complete destaining. Particular gel sectors are then transferred to distilled water and mounted on glass microslides for photography or for densitometric evaluation with a Leitz microspectrophotometer. These procedures have been used to identify characteristic differences in the electrophoretic mobilities of plasma enzymes and albumin fractions from several populations of poeciliid fishes. Observed differences in albumin mobilities (albumin phenotypes) were verified by mixing isoaliquots of test plasmas with plasma samples containing albumins of known mobility. Resultant patterns for albumin bands for such mixed plasmas were indistinguishable from those obtained with plasma samples from the F₁ hybrid progeny of parents possessing albumins of characteristically different electrophoretic mobilities. Procedural details for gel casting, electrophoresis and sample evaluation are described.     

Cascade stacking and cascade electrofocusing: their interconversion and fundamental unity.

The composition of a stack [an isotachophoresis (ITP) system] containing multiple trailing buffer constituents (“cascade stack”) was computed using a modification of the program of Routs. On electrophoresis, such a buffer mixture gave rise to multiple moving boundaries in which either buffer constituents or proteins could be stacked. Buffer zones within the stack served as “spacers.” The cascade stack exhibits a pH gradient, sharp zone boundaries, and constant zone width irrespective of the duration of electrophoresis, just as in the case of a stack comprising a single leading and trailing constituent. The pH gradient, sharp zone boundaries, and the sequential order of protein zones were maintained when the cascade stack was transposed between strongly acidic and basic electrolytes. Such a transposed isotachophoretic gel functioned as an electrofocusing system, indistinguishable from electrofocusing gels made in either buffers (buffer electrofocusing, or BEF) or Ampholine (isoelectric focusing, or IF). In the converse experiment, a cascade electrofocusing gel, formed in the same buffer mixture used to form a cascade stack, was subjected to electrofocusing until the steady-state was attained and then it was transposed between the appropriate upper and lower buffers of the corresponding cascade stacking system. Such transposition gave rise to moving zones with the typically sharp boundaries of a stack, a transient state pH gradient, and an order of protein zones within the cascade stack identical to the cascade electrofocusing system. These studies indicate the essential physical-chemical identity between these two types of electrophoretic systems and indicate the need for continued development of a unified theory for isoelectric focusing and steady-state stacking (isotachophoresis).     

Microheterogeneity of microtubule-associated proteins, MAP-1 and MAP-2, and differential phosphorylation of individual subcomponents

High molecular weight microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), prepared by copolymerization with tubulin, were electrophorectically separated into three and two major subcomponents, respectively, using 5% sodium dodecyl sulfate-polyacrylamide gels. By two-dimensional gel electrophoresis, all five MAP components were shown to possess a pI of around 5. Four of these proteins, MAP-1A, MAP-1C, MAP-2A, and MAP-2B, present in comparable amounts, were iodinated after electrophoretic separation and analyzed by two-dimensional peptide mapping. With both trypsin and V8 protease, almost identical patterns were obtained from MAP-2A and MAP-2B. MAP-1A and MAP-1C, too, gave similar digestion patterns, although some differences were noted. Incubation with [gamma-32P]ATP demonstrated that endogeneous protein kinase activities phosphorylated individual subcomponents at different rates. MAP-2A, the highest labeled component, was phosphorylated 2.5-fold compared to MAP-2B both in the presence and the absence of cAMP. Labeling of MAP-1 subcomponents was 4 times less than that of MAP-2A in the absence and 16 times less in the presence of cAMP. 32P-labeled MAP-2A and MAP-2B bands were indistinguishable by one-dimensional peptide mapping, as were the three MAP-1 bands. For both MAP-1 and MAP-2 subcomponents, cAMP induced phosphorylation at new molecular sites. Incubation of radiolabeled microtubule proteins with 1 mM ATP effected, upon electrophoresis, a clear shift of MAP-2A and MAP-2B bands to positions of higher apparent molecular weights, while only slightly affecting MAP-1 bands.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

Fractionation of Nondialyzable Melanoidin into Components by Electrofocusing Electrophoresis

Nondialyzable melanoidin formed in a model system of glucose and glycine was applied on thin layer gel electrofocusing in a polyacrylamide gel. The electrofocusing profile differed according to the reaction time: the pI of the melanoidin formed in the early stage of the reaction was less than 2.9 and, as the reaction progressed, the pI of the melanoidin formed gradually shifted to a less acidic value, from 2.9 to 3.3. Those from the xylose and glycine model system gave a similar profile to that of the glucose system.

Preparative separation of the nondialyzable melanoidin, which was formed by heating the glucose system for 7hr, was performed by flat-bed electrofocusing in Sephadex gel. At least 14 melanoidin bands were clearly electrofocused at a pH range of 2.7-3.3 and about 59% of the melanoidin applied remained unaffected by electrophoresis at the starting position. The major component of electrofocused melanoidin was pI 3.00, this being made up of 12.5% of the total amount of electrofocused melanoidin. The molecular weight of melanoidin affected by electrophoresis was about 25,000, regardless of the pI value of the melanoidin components. The reducing activity, estimated by the potassium ferricyanide method, showed that the lower the pI of melanoidin, the higher was the reducing activity.

The addition of hydrochloric acid to the melanoidin solution caused it to gradually become viscous and the melanoidin was precipitated below pH 3.0-3.5, corresponding approximately to ampholite. This feature can be used as a method to prepare nondialyzable melanoidin in a short time.