Purification and characterization of catabolic dehydroquinase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

Catabolic dehydroquinase which functions in the inducible quinic acid catabolic pathway in Neurospora crassa has been purified 8000-fold. The enzyme was purified by two methods. One used heat denaturation of contaminating proteins; the other used antibody affinity chromatography. The preparations obtained by these two methods were identical by all criteria. The purified enzyme is extremely resistant to thermal denaturation as well as denaturation 0y urea and guanidine hydrochloride at 25 degrees. It is irreversibly inactivated, although not efficiently dissociated, by sodium dodecyl sulfate and guanidine hydrochloride at 55 degrees. At pH 3.0, the enzyme is reversibly dissociated into inactive subunits. At high concentrations catabolic dehydroquinase aggregates into an inactive, high molecular weight complex. The native enzyme, which has a very high specific activity, has a molecular weight of approximately 220,000 and is composed of identical subunits of 8,000 to 12,000 molecular weight each. The native enzyme and the subunit are both asymmetric.     

Human adenine phosphoribosyltransferase. Affinity purification, subunit structure, amino acid composition, and peptide mapping.

Adenine phosphoribosyltransferase (EC 2.4.2.7) has been purified 55,000-fold from normal human erythrocytes. The native molecular weight of the enzyme is 38,200 as determined by sedimentation equilibrium centrifugation. The subunit molecular weight is 18,000 as determined by sodium dodecyl sulfate gel electrophoresis and 17,000 as determined by gel filtration in guanidine hydrochloride, suggesting that the enzyme is a dimer in its native state. Cross-linking the enzyme with dimethylsuberimidate confirms the dimeric structure and peptide mapping data suggested that the subunits are quite similar if not identical. The amino acid composition reveals that 33% of the residues are hydrophobic.     

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.     

Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties.

Kidney alkaline phosphatase was purified to homogeneity. It is a glycoprotein of about 172,000 molecular weight. Analyses of the subunit structure by sedimentation equilibrium in 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate indicate that the alkaline phosphatase is a dimer comprising two very similar or identical subunits of about 87,000 molecular weight. The native enzyme contains 4.5 +/- 0.2 g atoms of zinc per mol of protein. Reconstitution experiments from the apophosphatase show that binding of 4 Zn2+ per mol of dimer is essential for full activity. The kinetic data of Zn2+ binding to the apoprotein require at least a two-step mechanism, in which one of the steps corresponds to a conformational change within the enzyme. This paper also presents data concerning amino acid composition, sugar content, enzyme stability, absorbance index, and sedimentation velocity.     

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.     

Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.     

Physical properties of L-asparaginase from Serratia marcescens.

Purified L-asparaginase from Serratia marcescens had an apparent-weight average molecular weight of 171,000 to 180,000 as determined by electrophoresis on polyacrylamide gels and by sedimentation equilibrium at low speed in an analytical ultracentrifuge. A subunit molecular weight of 31,500 +/- 1,500 was estimated for the enzyme after treatment with sodium dodecyl sulfate and urea and electrophoresis on polyacrylamide gels; a similar value was obtained by high-speed sedimentation equilibrium in the presence of guanidine hydrochloride. Our data indicate that the Serratia enzyme could have five or six subunits of 32,000 daltons, compared to four subunits of 32,000 daltons in the Escherichia coli enzyme. The Serratia L-asparaginase also appears to be a larger molecule than the enzyme from Erwinia carotovora, Proteus vulgaris, Acinetobacter glutaminasificans, and Alcaligenes eutrophus. The Serratia enzyme, like that from E. caratovora, was more resistant than the E. coli enzyme to dissociation by sodium dodecyl sulfate. This resistance could be due to the finding that the Serratia enzyme had a relatively high hydrophobicity, similar to the enzyme from E. caratovora, when compared with the hydrophobicity of the E. coli enzyme. The isoelectric point of the Serratia enzyme was approximately 5.2. The influence of certain physical characteristics of the enzyme on the biological properties is discussed.     

The subunit structure of the cytochrome c oxidase complex.

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.     

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.     

Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.     

Nicotinamide deamidase from rabbit liver. 3. Inhibition and sedimentation studies

Treatment of rabbit liver nicotinamide deamidase with diisopropyl fluorophosphate or carbobenzoxyamide-2-phenylethyl chloromethyl ketone led to the loss of deamidase activity. In the process, two moles of reagent were covalently bound to the enzyme. Photo-oxidation of two histidine residues also led to the loss of deamidase activity. Acylated enzyme has been implicated as an intermediate in the hydrolysis of nicotinamide by the finding that nicotinamide hydroxyamate was formed when the reaction was carried out in the presence of hydroxylamine. These data are compatible with the characterization of the deamidase as a serine esterase.Guanidinium hydrochloride and sodium dodecyl sulfate, but not urea, caused the enzyme to dissociate. The molecular weight of the subunits obtained on dissociation with sodium dodecyl sulfate was shown to be 60,000 by electrophoresis in polyacrylamide gels. Nicotinamide deamidase was 70% inhibited by l-thyroxine at a molar ratio of thyroxine to enzyme of 2:1. Thyroxine caused the enzymes to enter into a rapid equilibrium involving dissociation into two subunits. The endogenous inhibitor of rabbit liver nicotinamide deamidase was found to be associated with the fatty-acid fraction and to have comparable effectiveness to equivalent weights of purified fatty acids.     

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.     

Renaturation of phosphorylase kinase activity from sodium dodecyl sulfate-polyacrylamide gels

Phosphorylase kinase activity is renatured and detected in situ following electrophoresis of the denatured holoenzyme in a sodium dodecyl sulfate-polyacrylamide gel containing phosphorylase b that has been included in the gel polymerization according to the method of R. L. Geahlen et al. [(1986) Anal. Biochem. 153, 151-158]. Among the enzyme's four subunits, only gamma is catalytically active. When extract of rabbit muscle is electrophoresed and renatured in a similar manner, the phosphorylase-conversion activity is also associated only with a protein band that comigrates with the gamma subunit of phosphorylase kinase. This suggests that the gamma subunit of phosphorylase kinase may be the sole activity in rabbit muscle responsible for the phosphorylation of phosphorylase b. In an alternative method for the renaturation of activity from conventional sodium dodecyl sulfate-polyacrylamide gels, the subunits of the enzyme are visualized using 2.5 M KCl, excised from the gel, and eluted by diffusion into buffer containing sodium dodecyl sulfate, which is subsequently removed by acetone precipitation of the eluted subunits. Catalytic activity is recovered when the acetone precipitate of the extracted gamma subunit is dissolved in 6 M guanidine hydrochloride and diluted 50-fold into an activity assay. Inclusion of eluted alpha and beta subunits in the assay inhibits the activity of the gamma subunit, which supports our previous finding that the alpha and/or beta subunits suppress the activity of the catalytic gamma subunit [H. K. Paudel and G. M. Carlson (1987) J. Biol. Chem. 262, 11912-11915].     

DNA-dependent RNA polymerases from Artemia salina. Subunit structure of polymerase II.

RNA polymerase II from larvae of the brine shrimp, Artemia salina, was highly purified by two cycles of DEAE-cellulose chromatography followed by centrifugation through discontinuous sucrose gradients. Gradient fractions were subjected to elctrophoresis is polyacrylamide gels containing sodium dodecyl sulfate. The subunit structure of RNA polymerase II was determined by quantitative comparison of the polypeptides and enzyme activity present in each gradient fraction. The enzyme contains one copy of each of four subunits with estimated molecular weights of 170,000, 130,000, 36,000 and 24,000. The total molecular weight agrees well with the molecular weight estimated for the native enzyme by density gradient centrifugation.     

Dissociation and reassembly of Escherichia coli type 1 pili.

Escherichia coli type 1 pili, which mediate the mannose-sensitive adherence of the bacterium to eucaryotic cells, are comprised of very stable arrays of pilin protein subunits (molecular weight, approximately 17,000). Previous methods for the dissociation of pili caused their irreversible denaturation. We have found that incubation of pili in saturated guanidine hydrochloride at 37 degrees C led to their complete dissociation, as evidenced by nephelometry and electron microscopy. Gel chromatography of the dissociated pili on a Sepharose CL-6B column in the presence of saturated guanidine hydrochloride yielded a single protein peak with a molecular weight corresponding to that of pilin. Dialysis of this peak against 5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) and rechromatography in the same buffer afforded a major protein peak, probably consisting of pilin dimers. About 25% of the protein in this peak bound to a mannan-sepharose column and could be eluted with methyl alpha-D-mannoside. The pilin dimer gave a single protein band upon polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (molecular weight, 16,600) or 10 M urea and penetrated completely into 7% gels in the absence of denaturants. Reassembly of the pilin dimers into pili was achieved upon dialysis against the tris(hydroxymethyl)aminomethane buffer containing 5 mM MgCl2, as observed by electron microscopy. Thus, the conditions used allow renaturation of the dissociated subunits and may aid in further studies of the structure-function relationship of pili.     

Epstein-Barr virus (EBV) antigenic determinants on subunits of the EBV determined nuclear antigen (EBNA)

In order to characterize the substructure of the Epstein-Barr virus determined nuclear antigen (EBNA) which is considered to have a molecular weight of 180 K in its native form, we have examined the antigenic specificity of the polypeptides obtained after denaturation of this molecule. Two procedures were employed; treatment by sodium dodecyl sulfate (SDS) and heat followed by gel electrophoresis, or denaturation by guanidine hydrochloride followed by gel filtration, which allowed us to detect a specific antigenic activity in the 50 K region, following dialysis. The denatured molecules could be reassociated into larger molecules (50 to 180 K) which retain the property of binding to fixed nuclei, as does native EBNA. These results indicate that EBNA has a polymeric structure and that 50 K subunits carry the antigenic determinants.     

Purification and preparation of antibody to RNA polymerase II stimulatory factors from Ehrlich ascites tumor cells.

An improved method was developed for purification of the protein termed S-II that specifically stimulates RNA polymerase II of Ehrlich ascites tumor cells. The specific activity of the final preparation was 400 000 units/mg of protein, which is about 30-fold higher than that of the previous preparation [Sekimizu, K., et al. (1976) Biochemistry 15, 5064]. The final preparation gave a single band on both sodium dodecyl sulfate and nondenaturing gel electrophoresis, and the protein extracted from the band on nondenaturing gel had stimulatory activity. S-II is a basic protein with a molecular weight of 40 500. The fundamental characteristics of S-II determined with the previous preparation were confirmed with completely purified S-II. A specific antibody to S-II was prepared. This antibody inhibited only the stimulatory activity of S-II and did not affect the activity of RNA polymerase II itself. Thus, S-II is probably not a component of the multimeric proteins of RNA polymerase II.     

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.     

Purification and catalytic properties of polygalacturonase isoforms from ripe avocado (Persea americana) fruit mesocarp

Endo-polygalacturonase (PG; EC 3.2.1.15) was recovered from the cell walls of avocado mesocarp ( Persea americana Mill cv. Lula) tissue and purified by sequential ion exchange and gel permeation chromatography. Two isoforms (S-I and S-II) were recovered, exhibiting molecular masses of about 41 kD on size exclusion media and about 48 (S-I) and 46 (S-II) kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both isoforms exhibited maximum activity at pH 6.0 against polygalacturonic acid (PGA) and hydrolyzed PGA of about 180 kDa to polymers of about 4 kDa. The catalytic activity of the 48-kDa isoform against PGA was slightly higher than that of the 46-kDa isoform. The purified PGs catalyzed significant molecular mass downshifts in the polyuronides of pre-ripe avocados; however, the capacity of the enzymes to solubilize polyuronides from cell walls of pre-ripe fruit was limited.     

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.