Physical properties and subunits of DNA dubia erythrocruorin.

The erythrocruorin of the freshwater leech Dina dubia possessed an S20,w of 61 S and exhibited a slightly sigmoid oxygenation curve with n congruent to 1.6 and P50 = 2.4 mm at pH 7.4. A minimum mol. wt of 23 000 +/- 2100 per heme group was determined from the iron and heme contents, 0.22 +/- 0.02 and 2.92 +/- 0.35 weight %. The subunit composition of this erythrocruorin was investigated using polyacrylamide gel electrophoresis and gel filtration in sodium dodecyl sulfate at neutral pH and gel filtration at pH 9. Sodium dodecyl sulfate electrophoresis of Dina erythrocruorin revealed the presence of five subunits (1-5) with mol. wts of about 13 000, 21 000, 23 000, 25 000 and 31 000, respectively. When the erythrocruorin was reduced with mercaptoethanol prior to dodecyl sulfate electrophoresis, three subunits (I-III) were observed, two possessing molecular weights in the range 12 000-14 000 (I and II) and one possessing a molecular weight of about 28 000. One of the subunits I, II, was provided by the dissociation of the 31 000 subunit. Subunit III (28 000) consisted of subunits 2, 3, and 4. It is likely that not all of the polypeptide chains constituting Dina erythrocruorin are associated each with a heme group.     

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subunits of Limnodrilus erythrocruorin.

The dissociation of the erythrocruorin of the oligochaete $\text{Limnodrilus gotoi}$ was investigated using polyacrylamide gel electrophoresis at neutral pH. In the presence of 0.1% SDS, the erythrocruorin dissociated into five subunits possessing molecular weights of 13,000 (1), 20,000 (2), 23,000 (3), 25,000 (4) and 47,000 (5). In the presence of SDS and mercaptoethanol, the erythrocruorin dissociated into two subunits, whose molecular weights were 13,000 (I) and 28,000 (II). Subunit I accounts for 70–80% of the whole molecule. SDS electrophoresis of the isolated subunits 1 through 5 in the presence of mercaptoethanol showed that subunit I was derived from both subunits 1 and 5, while subunit II was derived from subunits 2–4. These results suggest that $\text{Limnodrilus}$ erythrocruorin consists of at least five polypeptide chains: two chains of 13,000 and three chains of 28,000.     

Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes.

1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.     

Electron transfer mechanism and interaction studies between cytochrome C3 and ferredoxin

Ferredoxin, cytochrome c3 and hydrogenase are specific partners of the sulfate reduction pathway of Desulfovibrio desulfuricans Norway and might be exemplary for electron exchange mechanism studies. Cytochrome c3 contains four low redox potential haems for 13 000 molecular weight. Two ferredoxins isolated from the same bacteria are dimers of 6 000 molecular weight per subunit (Ferredoxin I: one (4 Fe-4S) cluster per subunit, ferredoxin II: two (4 Fe-4 S) clusters per subunit). The amino acid sequence of ferredoxin I is reported and compared to the ferredoxin II sequence. The structural characteristics of ferredoxins and cytochrome c3 should allow a discussion on the nature of the interaction. 1H-NMR spectra of ferredoxin I and cytochrome c3 in the absence and presence of ferredoxin are presented.     

Purification and subunit structure of RNA polymerases I and II from Dictyostelium discoideum vegetative cells

Summary A purification procedure to obtain RNA polymerases I (or A) and II (or B) from Dictyostelium discoideum amoeba has been developed. The enzymes were solubilized from purified nuclei and separated by DEAF-Sephadex chromatography. RNA polymerases I and II were further purified by a second chromatography on DEAE-Sephadex followed by chromatographies on phosphocellulose and heparin-sepharose. The specific activities of purified RNA polymerases I and II are 92 units/ mg protein and 70 units/ mg protein, respectively. The subunit structure of both RNA polymerases were analyzed by polyacrylamide gel electrophoresis under denaturing conditions after glycerol gradient centrifugation of the enzymes. The putative subunits of RNA polymerase I have molecular weights of 180 000,125 000,43 000,40 000,34 000, 31 000, 25 000,19 000, 17 000 and 14 000. The putative subunits of RNA polymerase II have molecular weights of 200 000 (170 000), 130 000, 33 000, 25 000, 19 000, 17 000, 15 000, 13 000. There are three polypeptides with common molecular weight in Dictyostelium RNA polymerases I and 11. The subunit of 25 000 daltons of both enzymes has common immunological determinants with RNA polymerase II from crustacean Artemia.Abbreviations TLCK tosyl-lysine-chloromethyl-ketone - DPT diazophenylthioether     

Preparation and characterization of monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris

Murine monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris were prepared by a modification of the method of Kohler and Milstein. 224 hybridomas were found to produce antibodies which bound to the hemoglobin; they were tested for binding to the four subunits of the hemoglobin: M (chain I, 16 kDa), D1 (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (50 kDa), a disulfide-bonded trimer of chains II, III and IV, each of about 17 kDa. 150 hybridomas bound to all four subunits and 40 hybridomas bound to various combinations of subunits. The remaining 34 hybridomas combined only with the hemoglobin. The twelve hybridomas obtained after subculturing and cloning were tested for their binding to the two fractions II and III, consisting of subunits D1 + D2 + T and M, respectively, obtained by dissociation at pH 9.5 and at pH 4.0 and to the reassociated whole molecules, obtained subsequent to return to neutral pH. Eight hybridomas which combined only with the hemoglobin also combined with all the reassociated molecules but not with any of the fractions: these monoclonal antibodies probably recognize conformation-dependent antigenic sites that are present only in the hexagonal bilayer structure characteristic of the native and reassociated hemoglobin molecules. Of the remaining four hybridomas, two bound to subunit T and two combined with subunits T and D2; they also combined with the reassociated molecules and with the fractions II. In addition, the hybridomas did not bind to the hemoglobins of Tubifex, Limnodrilus, Arenicola, Tylorrhynchus and Macrobdella or to the chlorocruorins of Myxicola and Eudistylia.     

5-Oxoprolinase from rat kidney, II. Molecular weight determinations.

The molecular weight of 5-oxoprolinase from rat kidney was estimated by gel filtration on Sephadex G-200 and G-150 to be 460 000 +/- 30 000. A value of 230 000 +/- 10 000 was obtained by zonal sedimentation in a sucrose gradient. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate yielded a molecular weightof 115 000 +/- 6 000. It is concluded that 5-oxoprolinase consists of four subunits of 115 000 daltons each. The dissociation or aggregation behavior of the enzyme seems to be influenced neither by the presence of the substrates 5-oxo-L-proline and MgATP2theta nor by the presence of the stabilizing compounds glutathione mercaptoethanol or dithioerythritol.     

A dodecamer of globin chains is the principal functional subunit of the extracellular hemoglobin of Lumbricus terrestris

Repeated dissociation of the approximately 3600-kDa hexagonal bilayer extracellular hemoglobin of Lumbricus terrestris in 4 M urea followed by gel filtration at neutral pH produces a subunit that retains the oxygen affinity of the native molecule (approximately 12 torr), but only two-thirds of the cooperativity (nmax = 2.1 +/- 0.2 versus 3.3 +/- 0.3). The mass of this subunit was estimated to be 202 +/- 15 kDa by gel filtration and 202 +/- 26 kDa from mass measurements of unstained freeze-dried specimens by scanning transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this subunit showed that it consists predominantly of the heme-containing subunits M (chain I, 17 kDa) and T (disulfide-bonded chains II-IV, 50 kDa). Mixing of subunits M and T isolated concurrently with the 200-kDa subunit resulted in partial association into particles that had a mass of 191 +/- 13 kDa determined by gel filtration and 200 +/- 38 kDa determined by scanning transmission electron microscopy and whose oxygen affinity and cooperativity were the same as those of the 200-kDa subunit. The results imply that the 200-kDa subunit is a dodecamer of globin chains, consisting of three copies each of subunits M and T (3 x chains (I + II + III + IV], in good agreement with the mass of 209 kDa calculated from the amino acid sequences of the four chains, and represents the largest functional subunit of Lumbricus hemoglobin. Twelve copies of this subunit would account for two-thirds of the total mass of the molecule, as suggested earlier (Vinogradov, S. N., Lugo, S. L., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8034-8038). The retention of only partial cooperativity by the 200-kDa subunit implies that full cooperativity is dependent on the presence of a complete hexagonal bilayer structure, wherein 12 200-kDa subunits are linked together by approximately 30-kDa heme-deficient chains.     

Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.     

Structure of beef heart mitochondrial F1-ATPase. Arrangement of subunits as disclosed by cross-linking reagents and selective labeling by radioactive ligands.

1. The following bifunctional reagents, dimethylsuberimidiate, dimethyladipimidate, methylmercaptobutyrimidate have been used to produce dimers between the neighboring subunits of beef heart F1-ATPase. 2. Treatment of beef heart F1-ATPase with dimethylsuberimidate or dimethyladipimidate resulted in the formation of four cross-linked products. Their molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 11 500, 105 000, 95 000 and 80 000, respectively. The products of molecular weight 115 000 and 105 000 were predominant and could be detected at the early stage of the cross-linking reaction. Treatment of beef heart F1-ATPase with methylmercaptobutyrimidate resulted in the accumulation of the product of molecular weight 115 000 and in traces of products of lower molecular weight. When the cross-linked products obtained with methylmercaptobutyrimidate were cleaved by beta-mercaptoethanol, the original gel electrophoresis pattern was restored. 3. Cross-linking of beef heart F1-ATPase by dimethylsuberimidate, dimethyladipimidate and methylmercaptobutyrimidate was accompanied by a loss of the ATPase activity. Cleavage of the cross-linked products obtained with methylmercaptobutyrimidate did not restore the original ATPase activity. 4. Identification of subunits A and B in the products of molecular weight 115 000 and 105 000 was achieved by specific labeling of subunit A with N-[14C]ethylmaleimide and of subunit B by chloronitro [14C]benzooxodiazole. Both products were able to bind N-[14C]ethylmaleimide; only the 105 000 dalton product was able to bind chloronitro [14C]benzooxodiazole. 5. The product of molecular weight 115 000 obtained by treatment of beef heart ATPase with methylmercaptobutyrimidate could bind N-[14C]ethylmaleimide. Its cleavage, following N-[14C]ethylmaleimide binding, yielded one labeled peptide identified with subunit A by polyacrylamide gel electrophoresis. 6. The above results indicate that the product of molecular weight 115 000 is a dimer containing two subunits A and that the product of molecular weight 105 000 is a dimer containing one subunit A and one subunit B. It can therefore be concluded that, in beef heart F1-ATPase, the A subunits are close to each other and that subunit A is close to subunit B. In contrast the B sublnits are probably too far from each other to be cross-linked by dimethylsuberimidate, dimethyladipimidate or methylmercaptobutyrimidate.     

The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality

The dissociation of the extracellular hemoglobin of Tubifex tubifex at alkaline and acid pH, and its reassociation upon return to neutral pH, was investigated using gel filtration, ultracentrifugation, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Tubifex hemoglobin dissociated at pH above 8 and below 6; both dissociations appeared to be equilibrium processes. The extent of dissociation increased as the pH moved away from neutrality; although dissociation was virtually complete at pH 11, its extent at acid pH did not exceed 50–60% at pH 4. Ca(II), Mg(II), and Sr(II) cations over the range 1–100 mm decreased the extent of the dissociation only at alkaline pH. The visible absorption spectrum of the oxyhemoglobin remained unaltered in the pH range 4–9. At more extreme pH, it changed with time, altering irreversibly to that of the aquo ferri form. Gel filtration of the hemoglobin at both extremes of pH showed that it dissociated into two heme-containing fragments; one consisting of subunit 1 (M_r ~ 17,000) and the other containing subunits 2, 3, and 4 of the hemoglobin (M_r ~ 60,000). Upon return to neutral pH, the dissociated fragment reassociated to the extent of 50 to 80% to whole hemoglobin molecules. The reassociation decreased with increase in alkaline pH, and with decrease in acid pH to which the hemoglobin had been exposed; it increased in the presence of Ca(II), Sr(II), and Mg(II) only subsequent to dissociation at alkaline pH. The SDS-PAGE patterns, gel-filtration elution volumes, and α-helical contents, determined from circular dichroism at 222 nm, of the reassociated whole molecules were identical to those of the native hemoglobin.     

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.     

Interaction of human serum apolipoprotein B with sodium deoxycholate

Preparation of apolipoprotein B (Apo B)-deoxycholate (DOC) complexes by gel filtration chromatography in the presence of 20 mM DOC, pH 8.5, gave two populations of particles with 5% (peak I) and 13% (peak II) lipid remaining bound. These complexes were initially shown to be very large and elongated, with partition radii of approx. 131 +/- 0.5 A, weight average molecular weights of approx. 164 000 +/- 1 000, and an intrinsic viscosity of 80.19 +/- 2.21 ml/g. Additionally, they appeared very similar to native low-density lipoprotein on sodium dodecyl sulfate-polyacrylamide gels, giving one major band. Incubation of these samples for 10 days under nitrogen at 4 degrees C in the presence of antibiotics and protease inhibitor resulted in dissociation to many smaller subunits. Results of scanning molecular sieve chromatography and analytical ultracentrifugation showed that dissociation of these complexes was relatively slow and indicated the presence of at least two classes of components in fresh samples: one a very elongated complex with a radius directly correlated to the DOC/Apo B ratio and inversely correlated to sample aging; and another of much smaller radius which was independent of DOC/Apo B ratio but directly correlated to sample aging; indicating that these dissociated subunits interact with each other to an appreciable extent. Furthermore, these complexes were found to undergo a preferential hydration upon interaction with DOC, which may contribute to large changes in their effective specific volumes, as well as to dissociation of subunits.     

Murine oncornavirus high-molecular-weight RNA structure thermal stepwise dissociation of 70S murine leukemia-sarcoma virus to subunits and low-molecular-weight associated RNAs.

The thermal dissociation into subunits and low-molecular-weight (LMW) associated RNAs of the aggregate structure of 70S RNA of a murine leukemia sarcoma viral complex was studied. By polyacrylamide-agarose gel electrophoresis, it was found that at low temperature a fraction of the genome was converted into an intermediate population of RNA (Im.P) with an apparent molecular weight of 6.6 times 10-6. At higher temperature, the 70S RNA and the Im.P RNA were successively dissociated into two RNA subunits called "I" and "II" and 70S-associated LMW RNAs. The apparent molecular weight of subunit I was about 5 times 10-6 and that of subunit II was about 3.2 times 10-6. The release of 4S, 5S, 5.5S, and 8S RNAs from 70S RNA at various temperatures was studied by composite polyacrylamide gel electrophoresis. It was found that the nature of hydrogen bonding to the 70S RNA was different for each LMW RNA species. A possible relationship of the association between the subunits and each 70S-associated LMW RNA, based on their T-m values, is discussed.     

Analysis of electrophoretic behavior of cytochrome c oxidase subunits. Retardation coefficient versus molecular weight in dodecyl sulfate urea gels.

1. Standard proteins were examined by electrophoresis in highly cross-linked polyacrylamide gels containing sodium dodecyl sulfate and urea. Their behavior was analyzed at a single gel concentration (by molecular weight vs. relative mobility) and at several gel concentrations (molecular weight vs. retardation coefficient). The validity of the latter method of analysis was established for this gel system. 2. Cytochrome c oxidase was subjected to analysis by this method. Compared to standards, subunits I and III showed increased free electrophoretic mobility, while that of subunit V was slightly decreased. The molecular weight values derived were: I, 44 600; II, 22 700; III, 23 500; IV, 16 900; V, 9400; VI, 7600; VII, 4300. The standard errors were all less than +/- 7%. 3. Isolated V and VI were analyzed by two dimensional dodecyl sulfate electrophoresis, in which the second dimension also contained urea. In contrast to their behavior when the holo-enzyme was examined, these isolated subunits V and VI no longer exchange migrating positions relative to each other during the two dimensional analysis. The molecular weight values of the isolated subunits agree with those of the holo-enzyme.     

Isozymes of alpha-galactosidase from Bacillus stearothermophilus

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.     

The mitotic apparatus. Physical chemical characterization of the 22S protein component and its subunits

The major 22S protein of the hexylene glycol-isolated mitotic apparatus has been characterized from spindle isolates and extracts of whole eggs and acetone powders of eggs from the sea urchins Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Arbacia punctulata. The protein is free of nucleotide, lipid, and ATPase activity. Essentially identical in amino acid composition, proteins from these species show a relatively high content of glutamic and aspartic acids and are fairly rich in hydrophobic amino acids. Optical rotatory dispersion studies indicate a helical content of about 20%, a value consistent with the proline content of the protein. The purified proteins have sedimentation rates in the range of 22-24S, diffusion constants of 2.4-2.5F, intrinsic viscosities of 3.7-4.3 ml/g, a partial specific volume of 0.74, and an average molecular weight of 880,000. Electron microscopy indicates a globular molecule with dimensions of approximately 150 by 200 A; such size and symmetry are consistent with hydrodynamic measurements. The 22S protein yields 6-7S, 9-10S, and 13-14S subunits below pH 4 or above pH 11. The 13-14S component has an estimated molecular weight of 600,000-700,000. A 5-6S particle is formed in 8 M urea or 5 M guanidine hydrochloride, while at pH 12 the 6-7S subunit is seen; each particle has a molecular weight of 230,000-240,000. In 8 M urea plus 2% mercaptoethanol or at pH 13, the molecular weight becomes 105,000-120,000; under these conditions the particle sediments at 2.5-3S and 4S, respectively. On the basis of these molecular weights, the 6-7S, 9-10S, 13-14S, and the parent 22S particle should be dimer, tetramer, hexamer, and octamer, respectively, of the 105,000-120,000 molecular weight subunit. The various subunits will reform the 22S particle when returned to neutral buffer, with the exception of the mercaptoethanol-treated urea subunit where breakage of disulfide bonds results in a polydisperse aggregate. The 22S particle itself is not susceptible to sulfhydryl reagents, implying either that the disulfide bonds are inaccessible or that they are unnecessary for maintenance of tertiary structure once the 22S particle has formed from subunits.     

Plant DNA-dependent RNA polymerases: subunit structures and enzymatic properties of the class II enzymes from quiescent and proliferating tissues

Class II DNA-dependent RNA polymerases were purified from soybean tissues of different physiological states: (1) from seed embryo tissue, representative of a quiescent, low metabolic state and (2) from auxin-treated hypocotyl tissue, representative of a highly proliferative and metabolically active state. Dodecyl sulfate, polyacrylamide gel electrophoresis indicates that RNA polymerase II from embryonic tissue consists largely (90-95%) of the form IIA enzyme, the largest subunit having a molecular weight of 215 000. RNA polymerase II from hypocotyl tissue is exclusively a form IIB enzyme, the largest subunit having a molecular weight of 180 000. Polypeptides common to RNA polymerases IIA and IIB have the following molecular weights: 138 000; 42 000; 27 000; 22 000; 19 000; 17 600; 17 000; 16 200; 16 100; and 14 000. Peptide mapping in the presence of dodecyl sulfate suggests that the 215 000 and 180 000 subunits possess similar peptide fragments. Plant embryo tissues do not contain protease activity capable of cleaving the 215 000 subunit to the 180 000 subunit, but proliferating plant tissues do contain such an activity. Mixing experiments indicate that appreciable amounts of RNA polymerase IIB are not being artifactually produced during protein purification.     

Chemical investigations on pig kidney aminoacylase.

1. Preparations of purified pig kidney aminoacylase (N-Acylamino-acid amidohydrolase, EC 3.5.1.14) were obtained by Sephadex and DEAE-cellulose chromatography in homogeneous form as judged by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. The apparent molecular weight of the enzyme, determined by gel filtration, was about 86 000. After treatment with mercaptoethanol, performic acid or sodium dodecyl sulphate a band with an apparent molecular weight of approximately 43 000 was observed in polyacrylamide gels containing sodium dodecyl sulphate. Thus pig kidney aminoacylase seems to be composed of two subunits. 3. The amino acid composition of the enzyme was determined. Aminoacylase contains 772 amino acids, which corresponds to a molecular weight of 85 500. 12 tryptophan and 12 half-cystine residues were found. 4. Each subunit of the enzyme contains two -SH groups of different reactivity and two disulfide bonds one of which is easily cleaved by -SH compounds, the second only by performic acid oxidation. 5. Chemical modification of two -SH groups abolishes the catalytic activity of aminoacylase. Cleavage of two disulfide bonds also inactivates the enzyme. It is suggested that the enzyme has two active sites each containing an essential -SH group and disulfide bond. One active site is assumed to be part of each subunit.