Proteins Released from Cell Envelopes of Pseudomonas aeruginosa on Exposure to Ethylenediaminetetraacetate: Comparison with Dimethylformamide-Extractable Proteins

Isolated cell envelopes of Pseudomonas aeruginosa were treated with N,N'-dimethylformamide (DMF) or with ethylenediaminetetraacetate (EDTA). DMF solubilized 73% of the dry weight of the cell envelope, 76% of the protein, 78% of the carbohydrate, and 76% of the phosphorus. Electron microscopy showed that DMF caused extensive alterations in the appearance of the cell envelope with blebs and bleblike vesicles predominating. After incubation with EDTA, the cell envelopes appeared to have lost material, but still retained the cell-like morphology. Analysis of DMF-solubilized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 16 protein bands. There were three major proteins that predominated, however, with molecular masses of 43,000 (protein A), 16,500 (protein B), and 72,000 daltons (protein C). Evidence is presented that protein A and protein B are glycoproteins. Gel electrophoresis of EDTA-solubilized material revealed that a number of proteins were released from the cell envelope. However, electrophoresis of an isolated protein-lipopolysaccharide complex released by EDTA showed that protein A and protein B were the major protein components of this complex. These data suggest that protein A and protein B are components of the outer cell wall membrane of P. aeruginosa. There is suggestive evidence that these proteins may play a role in maintaining the structural integrity of the cell envelope. Whether these proteins also have enzymatic activity could not be discerned from the present study, although it is possible that they may be associated with the terminal stages of lipopolysaccharide synthesis.     

Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

CHEMICAL CHARACTERIZATION OF ISOLATED EPIDERMAL DESMOSOMES

Desmosomes, isolated from cow nose epidermis by a method utilizing citrate buffer pH 2.6 and density gradient centrifugation, have been analyzed and found to contain approximately 76% protein, 17% carbohydrate, and 10% lipid. Nonpolar amino acids predominate in desmosomal protein, representing 456 residues per 1,000. The sialic acid content is 5 nM/mg of protein. The lipid fraction is composed of approximately 40% cholesterol and 60% phospholipids. Desmosomes are completely solubilized by incubation with 2% sodium dodecyl sulphate and 1% β-mercaptoethanol. Gel electrophoresis of the denatured desmosomal proteins reveals 24 bands, with mobilities corresponding to a molecular weight range of 15,000–230,000 daltons. Seven of these are considered to be major bands, together constituting 81% of the desmosomal protein. Bands 1 and 2, of molecular weights 230,000 and 210,000 daltons, together comprise 28% by weight of the desmosome. It is suggested that these protein chains are located in the desmosomal plaque. Bands 3 and 4 are PAS-positive, constitute 23% of the desmosomal protein, and have apparent molecular weights of 140,000 and 120,000 daltons, respectively. At least part of this material must originate from the carbohydrate-containing layer which is demonstrated, by histochemistry, to be present in the desmosomal interspace. The possible nature and origin of the remaining major bands, of molecular weights 90,000, 75,000, and 60,000 daltons, are discussed.     

Antibody inhibition of ferripyochelin binding to Pseudomonas aeruginosa cell envelopes

A 14K molecular weight protein which has been shown to bind ferripyochelin has been purified from cell envelopes of Pseudomonas aeruginosa low iron grown cells. The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to be free of contamination by lipopolysaccharide or carbohydrate. Antiserum to this protein was made in rabbits and was shown to react with the purified protein by immunoblot assay. The immunoglobulin G fraction of this antiserum blocked binding of [59Fe]pyochelin to isolated cell envelopes of P. aeruginosa in a dose-dependent fashion.     

Compositional characteristics of a chloroform/methanol soluble protein fraction from spinach chloroplast membranes.

Extraction of an aqueous suspension of spinach chloroplast lamellae with a chloroform/methanol mixture leads to solubilization of about 1/3 of the total membrane protein. Amino acid analysis of the chloroform/methanol-soluble protein shows that this fraction is largely enriched in the hydrophobic residues proline, leucine, alanine and phenylalanine and considerably depleted in polar amino acids, namely lysine and arginine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material reveals the presence of a variety of low molecular weight polypeptides (molecular weight less than or equal to 25 000), with more than 50% of the total fraction being contributed by a 25 000 dalton band. This band, which accounts for about 25% of the total chloroplast lamellar protein, has recently been identified as the main component of the light-harvesting chlorophyll-protein complex. The physiological role of most of the chloroform/methanol-soluble protein fraction is not known at present. From its chemical properties and apparent biological inertness, we propose that it plays mainly a structural role in situ, interacting with the lipid moiety of the chloroplast membrane. The material insoluble in the aqueous chloroform/methanol mixture is largely enriched in manganese, iron, cytochrome and water-soluble proteins, such as chloroplast coupling factor and ribulose diphosphate carboxylase.     

Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes.

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.     

The sodium channel from rat brain. Separation and characterization of subunits

Procedures are described for separation of the alpha, beta 1, and beta 2 subunits of the voltage-sensitive sodium channel from rat brain by gel filtration in sodium dodecyl sulfate (SDS) before and after reduction of intersubunit disulfide bonds or by preparative SDS-gel electrophoresis. Partial proteolytic maps of the SDS-denatured subunits indicate that they are nonidentical polypeptides. They are all heavily glycosylated and contain complex carbohydrate chains that bind wheat germ agglutinin. The apparent molecular weights of the separated subunits were estimated by gradient SDS-gel electrophoresis, by Ferguson analysis of migration in SDS gels of fixed acrylamide concentration, or by gel filtration in SDS or guanidine hydrochloride. For the alpha subunit, SDS-gel electrophoresis under various conditions gives an average Mr of 260,000. Gel filtration methods give anomalously low values. Removal of carbohydrate by sequential treatment with neuraminidase and endoglycosidase F results in a sharp protein band with apparent Mr = 220,000, suggesting that 15% of the mass of the native alpha subunit is carbohydrate. Electrophoretic and gel filtration methods yield consistent molecular weight estimates for the beta subunits. The average values are: beta 1, Mr = 36,000, and beta 2, Mr = 33,000. Deglycosylation by treatment with endoglycosidase F, trifluoromethanesulfonic acid, or HF yields sharp protein bands with apparent Mr = 23,000 and 21,000 for the beta 1 and beta 2 subunits, respectively, suggesting that 36% of the mass of the native beta 1 and beta 2 subunits is carbohydrate.     

On the relationship between water-soluble protein and calcium in the egg ofOryzias latipes

Summary Quantitative analysis of total and bound calcium in the egg ofOryzias latipes was performed during; the course of early development. The amount of total calcium is nearly constant. 95% of total calcium is bound with the basic substance before fertilization, but 17% becomes free within 10 minutes after fertilization. The rate of bound calcium to total calcium recovers the original value by the blastula stage. The water-soluble protein patterns obtained by paper electrophoresis consist of five anion bands. The protein of band IV increases during the early embryogenesis, while that of band I decreases relatively. There are no remarkable changes in the amount of protein in bands II, III, and V. Among three bands of thin layer chromatogram with Sephadex gel (G-200), the amount of protein of band A (more than 160,000 in molecular weight) reduces gradually after fertilization, while no marked change is observed in bands B (about 100,000 in molecular weight) and C (about 30,000 in molecular weight).The greater part of incorporated⁴⁵Ca is detected in low molecular substance (less than 30,000 in molecular weight), and the rest in bands A, B, and C, respectively.     

M BAND PROTEIN : Two Components Isolated from Chicken Breast Muscle

M band protein can be specifically extracted from fresh chicken breast muscle myofibrils suspended in 5 mM Tris-HCl pH 8.0. During discontinuous polyacrylamide gel electrophoresis the isolated protein separates into three bands which can be identified as two separate components (A, B) and a complex of the two. When partially purified fractions of the separated components are combined, an increase in the intensity of the band containing the complex can be shown. The polypeptide chain weights of the two components are 100,000 (A) and 40,000 (B) daltons as estimated by sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Antibody prepared against total M band protein stains only the M band of the myofibril and is completely absorbed by M band protein. M band protein also absorbs the M band staining specifically from antibody which stains both I and M bands. Immunodiffusion data indicate that anti-M band is a mixture of two specific antibodies, one against each component.     

Studies on (K+ + H+)-ATPase V. Chemical composition and molecular weight of the catalytic subunit

(1) A (K+ + H+)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The Detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH2 terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, N = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S. E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

Identification of a nonimmunoreactive but highly bioactive form of prolactin in the mouse pituitary by gel electrophoresis.

Polyacrylamide gel electrophoresis of fresh mouse pituitary extracts in 10% acrylamide reveals three closely situated bands of protein in the area where prolactin usually migrates. The fastest migrating band constitutes only 10–30% of the total, but it is twice as active in the pigeon crop-sac bioassay as the major band and has little or no immunologic cross-reactivity against an antiserum to the major constituent. This newly recognized band may be a hitherto unrecognized molecular variant of prolactin.     

Protein and Carbohydrate Composition of the Cell Envelope of Halobacterium salinarium

The isolated cell envelope of Halobacterium salinarium strain 1 contained 15 to 20 proteins that were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All but one of these proteins had molecular weights of 130,000 or less and together accounted for 50 to 60% of the total envelope protein. The remaining 40 to 50% of the envelope protein was accounted for by a single protein with an apparent molecular weight of approximately 194,000 that stained for carbohydrate with periodate-Schiff reagent. The proteolytic enzymes trypsin and Pronase were used to show that the carbohydrate is covalently bound to the protein. Separation of amino sugar- and hexose-containing tryptic peptides by gel filtration indicated that all of the nonlipid carbohydrate of the cell envelope is covalently bound to protein. The results of partial purification by phenol extraction indicated that both the amino sugar and hexose are bound to the 194,000-molecular-weight protein. Exposure of isolated cell envelopes to low salt concentration resulted in solubilization of a majority of the envelope proteins. A relatively small number of proteins, including the high-molecular-weight, carbohydrate-containing protein, remained bound to the sedimentable cell membrane fraction.     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Carbohydrate composition of sarcolemma of rat myocardium

The carbohydrate composition of SL prepared from rat ventricular muscle was examined and compared with those of Mw and FSR prepared from the same species. The total carbohydrate content of SL was 308.58 micrograms/mg of protein, which was greater than that of Mw (74.49 micrograms/mg of protein) and FSR (76.50 micrograms/mg of protein). The carbohydrate of SL was composed of hexose (35.0%), hexuronic acid (7.3%), sialic acid (6.1%), methyl pentose (7.4%), and hexosamine (44.2%). The peculiar PAS-positive glycoproteins of SL were observed as five bands on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and the molecular weight of the main band of these was 85.4 kDa. Thus, each of the cell fractions obviously shows a distinct carbohydrate composition and the results obtained in the present study may suggest that the presence of the PAS-positive glycoprotein (85.4 kDa) can be used for routine monitoring of the purity of the SL fraction.     

Study of envelope proteins in E. coli cet and recA mutants by SDS acrylamide gel electrophoresis

Disc-electrophoresis of E. coli envelope proteins on SDS acrylamide gels reproducibly revealed up to 50 distinct polypeptide bands. Corresponding molecular weights ranged from 105,000 to 20,000 daltons or less. Major bands corresponded to molecular weights of 73,000, 48,000, 36,000 and 30,000 with the latter constituting up to 20% of the total envelope protein depending upon the method of isolation. Minimum levels of detection using stained gels equaled 0.25 μg protein or 1% of total sample analyzed; for a polypeptide of molecular weight 40,000 daltons this was calculated to be equivalent to 1,200 molecules per cell envelope. In envelopes from a cetB^? mutant strain (refractory to colicin E2), an additional band, constituting up to 5% of the total envelope protein was present. The molecular weight of this protein, which was maximally present in wild type envelopes in only trace amounts, is 44,000 daltons, indicating a cellular concentration of approximately 6 × 10³ molecules per envelope. This new band was not affected by heating envelope preparations to 100° prior to electrophoresis, but was largely eliminated by washing isolated envelopes in low ionic strength buffer, or by pre-incubating cells with trypsin prior to preparation of envelopes. Treatment of isolated envelopes with Triton X-100, which preferentially releases inner membrane proteins from the envelope (18), resulted in the extraction of a preponderance of the high molecular weight polypeptides, including the 44,000 dalton protein from envelopes of the mutant. The major polypeptides of the envelope and the low molecular weight components were not extracted by Triton X-100. The properties of the 44,000 dalton protein indicated that it is relatively loosely associated with the surface envelope and may be exposed on the external surface of the cytoplasmic membrane. Possible explanations for the appearance of this protein in mutant strains and its relationship to the inability of these to respond, specifically to surface bound colicin E2, will be discussed. Extensive analysis of envelopes from recA^? mutants was also carried out and revealed an unusual amount of variation in polypeptide profiles obtained from different preparations. However, no consistent quantitative or qualitative difference between recA and rec⁺ strains was obtained. In recA, cetB double mutants, the increased level of the 44,000 dalton polypeptide was identical to that found in the rec⁺, cetB mutant.     

Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes.     

Outer membrane proteins of Escherichia coli. I. Effect of preparative conditions on the migration of protein in polyacrylamide gels

Outer membrane protein of Escherichia coli prepared for polyacrylamide gel electrophoresis by solubilization of the membrane in an organic solvent followed by dialysis into sodium dodecyl sulfate (SDS) solution or by solublization of the membrane directly in SDS solution followed by dialysis into a SDS-urea solution and brief heating at 100 °C resulted in a simple polypeptide profile on SDS-containing gels. This polypeptide pattern was characterized by a single major protein band migrating with an apparent molecular weight of about 42,000 daltons which accounted for about 70% of the total protein on the gel. However, if the outer membrane protein is dissolved in SDS solution without urea and heated at 70 °C, major bands are observed in three regions of the gel: A broad band or group of bands near the top of the gel with an apparent molecular weight of much greater than 42,000 daltona (peak A), a second band with the same mobility as the 42,000-dalton band in boiled samples (peak B), and a third, faster-migrating band with an apparent molecular weight of less than 42,000 daltons (peak C).Elution of protein from A or C followed by heating at 100 °C converts this protein to a form migrating with peak B. If the outer-membrane protein is dissolved in SDS solution at 37 °C with no further heating and applied to gels, peak B dissappears completely and A and C increase. These can be partially converted to peak B by urea treatment. Protein from peaks A and C was isolated by chromatography on Sephadex in the presence of SDS, and the intrinsic viscosity of this protein was measured before and after boiling. The intrinsic viscosity of protein from peak A was 35 cc/g both before and after boiling, while the intrinsic viscosity of protein from peak C was 28 cc/g before boiling and 35 cc/g after boiling. These results are best explained by assuming that the protein in peak A represents aggregates of a 42,000-dalton species which are dissociated by boiling or solvent treatment and that the protein in peak C represents a monomeric form of the 42,000-dalton protein which is not fully reacted with SDS and which is converted to the “rigid rod” conformation characteristic of protein-SDS complexes only upon boiling or solvent treatment.     

The Isolation and Characterization of Plasma Membranes From Calf Thymocytes

A simple method is described for the isolation and characterization of plasma membranes from calf thymocytes. The procedure involves extraction of thymocytes in a hypotonic medium containing borate and EDTA. Membrane ghosts, obtained by centrifugation of the cell lysate, are purified by passage through a column containing glass beads. The purity of plasma membranes was checked by chemical analysis, by assay of marker enzymes and also by electron microscopy. Polyacrylamide gel electrophoresis of the calf thymocyte plasma membrane produced a number of protein bands as well as a major band which stained for carbohydrate. The method is rapid and could be applied to isolate plasma membranes from nucleated cells of various types in large quantities.     

The isolation and characterization of plasma membranes from calf thymocytes.

A simple method is described for the isolation and characterization of plasma membranes from calf thymocytes. The procedure involves extraction of thymocytes in a hypotonic medium containing borate and EDTA. Membrane ghosts, obtained by centrifugation of the cell lysate, are purified by passage through a column containing glass beads. The purity of plasma membranes was checked by chemical analysis, by assay of marker enzymes and also by electron microscopy. Polyacrylamide gel electrophoresis of the calf thymocyte plasma membrane produced a number of protein bands as well as a major band which stained for carbohydrate. The method is rapid and could be applied to isolate plasma membranes from nucleated cells of various types in large quantities.     

Studies on (K + H)-ATPase. V. Chemical composition and molecular weight of the catalytic subunit

(1) A (K⁺ + H⁺)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH₂ terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, n = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.