Specific interaction of the tetragonally arrayed protein layer of Bacillus sphaericus with its peptidoglycan sacculus.

Tetragonal layer protein (T-layer) isolated from Bacillus sphaericus NTCC 9602 (wild type) or 9602 Lmw (variant) bonded specifically to the sacculi (peptidoglycan) of either cell type. Only uncleaved T-layer subunits were capable of specific recognition of the B. sphaericus sacculi; other Bacillus strains and gram-positive bacterial sacculi would not adsorb B. sphaericus strain 9602 T-layer. The peptidogylcan did not function as a template since isolated T-layer subunits self-assembled into characteristic pattern. Upon reassociation with sacculi, T-layer assemblies were randomly oriented patches compared with more continuous strictly oriented pattern on cells or fresh cell walls. T-layer associated with the sacculus was less susceptible to conditions that dissociated in vitro-assembled T-layer. Mild proteolysis of both wild-type and variant T-layer subunits by a variety of enzymes reduced the molecular weight by 18,000 in all cases, indicating that one region of the molecule was particularly susceptible to cleavage. Subunits from which the minor fragment had been cleaved upon aging retained the capacity to assemble in vitro, but would no longer adsorb to sacculi. Thus, the ability of T-layer to form networks was separate from its ability to bind cell walls, and the 18,000-dalton piece of the T-layer polypeptide was necessary for attachment to the cell wall.     

Heterogeneity of delta-crystallins of the embryonic mallard lens. Correlation between subunit compositions and isoelectric points.

delta-Crystallins from the lenses of embryonic mallards (Anas platyrhynchos) were analyzed with respect to native and subunit molecular weight, subunit composition, and isoelectric point. NaDodSO4-urea-polyacrylamide gel electrophoresis showed that unfractionated mallard delta-crystallins are composed of approximately equal amounts of subunits with molecular weights near 47 000 and 48 000. Agarose gel chromatography showed that the embryonic mallard delta-crystallins have native molecular weights slightly less than 200 000. Thus, embryonic mallard delta-crystallins appear to be tetramers. Five major and nine minor delta-crystallins were resolved by isoelectric focusing. The five predominant delta-crystallins each cross-reacted with antichick delta-crystallin antiserum, and each had a different proportion of the larger and smaller subunits, indicating a direct relationship between the isoelectric point and the subunit composition. The presence of numerous, minor species of native delta-crystallins with different isoelectric points suggested that the subunits possess charge heteogeneity as well as size heterogeneity.     

RAPID CONCURRENT AUTOMATED FLUORIMETRIC ASSAY OF NORADRENALINE, DOPAMINE, 3, 4-DIHYDROXYPHENYLACETIC ACID, HOMOVANILLIC ACID AND 3-METHOXYTYRAMINE IN MILLIGRAM AMOUNTS OF NERVOUS TISSUE AFTER ISOLATION ON SEPHADEX G10

Abstract— Brain tubulin subunits were separated by a combination of isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a two-dimensional polyacrylamide slab gel technique. Isoelectric focusing separated tubulin subunits into two major groups of bands, such that the more acidic group corresponded to the α subunit and the less acidic group corresponded to the β subunit. In addition, isoelectric focusing resolved the β subunit into two subspecies which differed slightly in isoelectric properties but were the same apparent molecular weight. The a subunit was resolved into many subspecies that appear to differ from each other by both apparent molecular weight and isoelectric properties.     

Chemical characterization of the regularly arranged surface layers of Clostridium thermosaccharolyticum and Clostridium thermohydrosulfuricum.

Clostridum thermosaccharolyticum and Clostridium thermohydrosulfuricum possess as outermost cell wall layer a tetragonal or hexagonal ordered array of macromolecules. The subunits of the surface layer can be detached from isolated cell walls with urea (8M) or guanidine-HCl (4 to 5 M). Triton X-100, dithiothreitol, ethylenediaminetetracetate, and KCl (3 M) had no visible effect on the regular arrays. Sodium dodecyl sulfate-polyacrylamide electrophroesis showed that, in both organisms, the surface layer is composed of glycoprotein of molecular weight 140,000. The glycoprotein from both microorganisms has a predominantly acidic amino acid composition and an acidic isoelectric point after isoelectric focusing on polyacrylamide gels. The glycocomponent is composed of glucose, galactose, mannose, and rhamnose.     

Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica

The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

Localization, isolation and characterization of Mn-superoxide dismutase in bovine adrenocortical cells

Cyanide-insensitive superoxide dismutase activity was present in both cytosol (26%) and mitochondrial (64%) fractions of bovine adrenal cells. The cyanide-insensitive superoxide dismutase was isolated from the mitochondrial fraction. It contained 2.2 g atoms of manganese per mol of enzyme. The enzyme had a molecular weight of 82,000 and a subunit molecular weight of 22,000. The isoelectric point, amino acid composition, and spectra of visible and ultraviolet regions were similar to those of the Mn-superoxide dismutase from the other mitochondria.     

Subunit structure of Achromobacter collagenase.

The highly active form of collagenase (EC 3.4.24.3) from Achromobacter iophagus (specific activity 2 microkat/mg) has a molecular weight of 70,000 and the sedimentation coefficient s20,2 = 4.4 S. It is composed of two subunits of molecular weight 35,000 and s20,w of 2.9 S. The dissociation of the dimer under different conditions resulted in the complete and irreversible loss of enzymic activity. A unique N-terminal sequence Thr-Ala-Ala-Asp-Leu-Glu-Ala-Leu-Val- indicates that the two subunits are identical, at least in the N-terminal part of the polypeptide chain. Reduction and pyridylethylation of the subunit change neither molecular weight nor amino acid composition: therefore each subunit of molecular weight 35,000 consists of a single polypeptide chain. Another active and homogeneous form of Achromobacter collagenase (specific activity 1.64 microkat/mg) gives a value for the apparent molecular weight of 80,000 on sodium dodecyl sulphate-polyacrylamide electrophoresis. It is also a dimer in which each of the two subunits of molecular weight 35,000 binds non-covalently a peptide of molecular weight 5000. The dissociation of this form of collagenase is also accompanied by irreversible loss of enzymic activity. The amino acid composition of the subunits which were isolated from both 70,000 and 80,000 collagenases is the same. The role of dimer-monometer equilibrium in the biological function of collagenase is discussed.     

P61 Protein from a Male Sterile Mutant of Rice is an Isoform of the Chloroplast ATPase β Subunit

P61 was a protein identified from chloroplasts of Nongken 58S, a male sterile mutant of rice (Oryza sativa L. ssp. japonica). Microsequence analysis has revealed that its N-terminal sequence was identical to N-termini of ATPase β subunits of chloroplasts from rice and barley. The antiserum produced using ATPase β subunit from maize specifically recognized P61. P61 had the same molecular weight as the chloroplast ATPase β subunit of wild-type rice “Nongken 58”, but had different isoelectric point (pI) from this β　subunit. P61 was more basic than this β subunit. Thus, P61 would be identified as an isoform of the chloroplast ATPase β subunit of rice, named β1. Genetic analysis with a F2 population of Nongken 58SדNongken 58” showed that a single recessive genic gene regulated the formation of β1.     

Analysis of the dominance of mutations in cAMP-binding sites of murine type I cAMP-dependent protein kinase in activation of kinase from heterozygous mutant lymphoma cells.

Structural lesions in cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase caused identical increases in apparent constants for cyclic nucleotide-dependent kinase activation in preparations from cells that were hemizygous or heterozygous for mutant R1 subunit expression. No wild-type kinase activation was observed in extracts from heterozygous mutant cells. This "dominance" was investigated by characterizing expression of wild-type and mutant R1 subunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild-type R1 subunits and R1 subunits with a lesion (Glu200) that inactivates cAMP-binding site A. By both studies of cAMP dissociation and two-dimensional gel analysis, wild-type R subunits comprised about 35% of total R1 subunits in heterozygous mutants. Synthesis of wild-type and mutant R1 subunits was equivalent, but wild-type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel R1 subunit-containing species from heterozygous mutant preparations whose elution behavior suggested a trimeric kinase consisting of an R1 subunit dimer and one catalytic (C) subunit. Wild-type R1 subunit was found only in dimer and "trimer" peaks; the tetrameric kinase peak contained only mutant R1 subunit. It is concluded that C subunit binds preferentially to mutant R1 subunit in heterozygous cells forming either tetrameric kinase with mutant R1 subunit homodimers or trimeric kinase with R1 subunit heterodimers. This preferential binding results both in suppression of wild-type kinase activation and differential stabilization of mutant R1 subunits.     

Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits.

Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) from fresh human erythrocytes has been purified to homogeneity in two steps with an overall yield of 56%. The purification involves DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharose/formycin B. This scheme is suitable for purification of the phosphorylase from as little as 0.1 ml of packed erythrocytes. The native enzyme appears to be a trimer with native molecular weight of 93,800 and the subunit molecular weight of 29,700 +/- 1,100. Two-dimensional gel electrophoresis of the purified enzyme under denaturing conditions revealed four major separable subunits (numbered 1 to 4) with the same molecular weight. The apparent isoelectric points of subunits 1 to 4 in 9.5 M urea are 6.63, 6.41, 6.29, and 6.20, respectively. The different subunits are likely the result of post-translational modification of the enzyme and provide an explanation of the complex native isoelectric focusing pattern of purine nucleoside phosphorylase from erythrocytes. Three of the four subunits are detectable in two-dimensional electrophoretic gels of crude hemolysates. Knowing the location of the subunits of purine nucleoside phosphorylase in a two-dimensional electropherogram allows one to characterize the purine nucleoside phosphorylase in crude cell extracts from individuals with variant or mutant purine nucleoside phosphorylase as demonstrated in a subsequent communication. Partial purification of the phosphorylase from 1 ml of erythrocytes on DEAE-Sephadex increases the sensitivity of detection of the subunits to the 0.3% level.     

Purification and characterization of gamma-glutamyl transpeptidase from human pancreas

Gamma-glutamyl transpeptidase was purified from human pancreas to an electrophoretically homogeneous state. The enzyme was separated into two active fractions on a DEAE-cellulose column. Both enzyme preparations had the same molecular weight (9 x 10(4)) and were composed of two nonidentical subunits (molecular weight, 61,000 and 27,000). While the optimum pH and pH- and thermal-stability range of both enzymes were identical, their isoelectric points were considerably different. Following incubation with neuraminidase, however, the isoelectric point of F-11 became similar to that of F-I, suggesting that this difference in electrophoretic mobility is due to a difference in the content of sialic acid moiety.     

Further characterization of L-beta-hydroxyacid dehydrogenase from Drosophila

L-beta-Hydroxyacid dehydrogenase (L-beta-hydroxyacid-NAD-oxidoreductase, EC 1.1.1.45) of Drosophila is composed of two, identical subunits with a molecular weight of approx. 33 300. The enzyme was purified 938-fold from Drosophila melanogaster. An isoelectric point of 8.6 was determined for L-beta-hydroxyacid dehydrogenase. An amino acid analysis was conducted of the purified enzyme. A single subunit was obtained by SDS-gel electrophoresis of the purified enzyme. Translation of larval and adult mRNA in a mRNA-dependent reticulocyte lysate, followed by immune precipitation using anti-L-beta-hydroxyacid dehydrogenase IgG revealed a single L-beta-hydroxyacid dehydrogenase subunit of 33 300. Larval and adult proteins were the same size. The enzyme does not appear to be subjected to substantial post-translational modifications.     

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.     

Electron microscopic study of the interaction between phages and Bacillus thuringiensis cells

The interaction of phages belonging to different morphological groups with the cells of Bacillus thuringiensis var. galleriae R and S variants was studied. No adsorption of phages Tg11 and Tg18 on the cells of R variant was found upon infection in a liquid medium. What is characteristic of phage Tg11 is that it is predominantly adsorbed at the poles of S variant cells. Phage Tg18 particles are uniformly distributed along the perimeter of S variant cells. Phage Tg13 is adsorbed on the both variant cells. Phage aggregates with the elements of cell walls having a tetrahonal assembly of the subunits can be revealed in phage Tg13 lysates. The size of the subunits is 7 nm and the distance between their centers is 11 nm. A structured element, apparently the T-layer, is involved in the adsorption of phage Tg13 on the cells.     

Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Purification and some properties.

The purine nucleoside phosphorylases from Escherichia coli and from Salmonella typhimurium have been purified to electrophoretic homogeneity and crystallized. Comparative studies revealed that the two enzymes are very much alike. They obey simple Michaelis-Menten kinetics for their substrates with the exception of phosphate for which they show negative cooperativity. Gel filtration on Sephadex G-200 of the native enzymes revealed a molecular weight for both enzymes of 138000 plus or minus 10%. By use of dodecylsulphate gel electrophoresis a subunit molecular weight of 23700 plus or minus 5% was determined, suggesting that both enzymes consist of six subunits of equal molecular weight. When the subunits were partially crosslinked with dimethyl suberimidate before dodecylsulphate electrophoresis six protein bands were observed in agreement with the proposed oligomeric state of the enzyme, consisting of six subunits of equal molecular weight. Analysis of the amino acid composition also indicates that the subunits are identical. 6M guanidinium chloride dissociates the enzymes; association experiments with native and succinylated enzymes suggested that only the hexameric form is active. Both enzymes could be dissociated into subunits by p-chloromercuribenzoate; this dissociation is prevented by the substrates: the nucleosides, the pentose 1-phosphates, and mixtures of phosphate and purine bases.     

A chemical and physical comparison of ferritin subunit species fractionated by high-performance liquid chromatography

Selected chemical and physical properties were measured for different forms of ferritin subunits which had been separated by reverse-phase high-performance liquid chromatography. Ferritin subunits from porcine spleen behaved, on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, as though they were ~ M_r 2000 larger than equine spleen ferritin, whereas no difference in size was observed by gel chromatography in 6 m guanidinium chloride. All subunit species exhibited similar isoelectric focusing properties. In contrast to previous reports, no carbohydrate could be found associated with any of the isolated subunit species. Thus, the aberrant behavior of the porcine ferritin subunits between the two empirical molecular weight estimation methods appears to be the result of factor(s) other than protein intrinsic charge or covalently attached carbohydrate.     

Purification, properties, and crystallographic data for a principal nontoxic lectin from seeds of Abrus precatorius.

Nontoxic abrus lectin has been prepared by a new purification procedure. The method is accomplished by 45% saturation ammonium sulfate fractionation from a 5% acetic acid extract of the seeds of Abrus precatorius followed by diethylaminoethyl-Sephadex A-50 and Sepharose 4B affinity chromatography. The abrus lectin appeared homogeneous as judged by electrophoresis, analytical ultracentrifugation, and isoelectric focusing. The lectin molecule has a weight of 126,000 as determined by sedimentation equilibrium. It is composed of four subunits, of which two pairs have either identical or closely similar molecular sizes (33,800 and 32,200 daltons) as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of amino acid analyses are given; none of the cysteic acid appears to arise from cysteine. An electrofocusing experiment indicated the isoelectric point to be 5.0. Crystals large enough for x-ray investigation were obtained by a vapor diffusion technique. X-ray precession photographs revealed that abrus lectin crystallizes in a tetragonal unit cell of symmetry P41212 and dimensions a = 140 and c = 210 A. The asymmetric unit contains 2 protein molecules of molecular weight 126,000 and has a solvent content of approximately 41% by volume.     

Psychrophilic, mesophilic, and thermophilic triosephosphate isomerases from three clostridial species.

Triosephosphate isomerase was purified to homogeneity as judged by analytical gel electrophoresis from clostridium sp. strain 69, clostridium pasteurianum, and C. thermosaccharolyticum, which grow optimally at 18, 37, and 55 C, respectively. Comparative studies on these purified proteins showed that they had the same molecular weight (53,000) and subunit molecular weight (26,500). They were equally susceptible to the active site-directed inhibitor, glycidol phosphate. However, their temperature and pH optima, as well as their stabilities to heat, urea, and sodium dodecyl sulfate, differed. The proteins also had different mobilities in acrylamide gel electrophoresis. This difference in ionic character was also reflected in the elution behavior of the enzymes from hydroxyapatite and in the isoelectric points determined by isoelectric focusing in acrylamide gel. The amino acid composition of these proteins showed that the thermophilic enzyme contains a greater amount of proline than the other enzymes. The ratio of acidic amino acids to basic amino acids was 1.79, 1.38, and 1.66 for the thermophilic mesophilic and psychrophilic enzymes, respectively. This is consistent with the relative isoelectric point values of these three enzymes.     

Evidence for a multiple subunit composition of plant NAD malic enzyme

Malate dehydrogenase (decarboxylating) (EC 1.1.1.39) was purified to near homogeneity from both a C3 plant, Solanum tuberosum, and a CAM plant, Crassula argentea. Sodium dodecyl sulfate-gel electrophoresis of both enzymes revealed an alpha,beta subunit composition with corresponding molecular mass assignments of 61,000 and 55,000 daltons. Isoelectric focusing under native conditions showed only one constituent malic enzyme form with an isoelectric point of 5.1. No evidence of additional isoenzymes was found. Urea isoelectric focusing showed the alpha subunit to be more acidic than the beta subunit. Peptide mapping by limited proteolysis with Staphylococcus aureus V-8 protease, trypsin, and endoproteinase Arg-C eliminated the possibility that a precursor-product relationship may have existed between the two subunits and demonstrated that they each possess unique primary sequences. Further support for this conclusion was obtained when significant differences in the contents of glutamic acid, isoleucine, and arginine were revealed by amino acid analysis of the isolated subunits. There was no apparent activity associated with the separated subunits (as resolved by urea-DEAE chromatography), but activity could be found in a reconstituted system, thereby indicating an (alpha,beta)n protomeric configuration. This is the first case where malic enzyme has been conclusively shown to be constructed from nonidentical subunits. This phenomenon has been observed only for the NAD malic enzyme isolated from plants.