Studies of soluble rat liver mitochondrial acid ATPases: II. Structural and immunological properties of ATPase 1

We described previously the existence of a soluble ATPase activity in rat liver mitochondria [1]. The purification and catalytic properties have been described [2]. In a continuation of these experiments, we have studied the immunologic and structural properties of one molecular form of this enzyme: ATPase I.We have prepared the antiserum anti-ATPase I and demonstrated the purity of our enzyme preparation by immunodiffusion and immunoelectrophoresis. An immunohistochemical method also confirmed the localization of ATPase I in the soluble fraction of mitochondria.The molecular weight of ATPase I was measured by G 100 Sephadex gel filtration and was found to be 18,400; electrophoresis on polyacrylamide gels gave a value of 18,600. The pHi of ATPase I was found to be 7,2.Amino acid analysis showed high amounts of aspartic acid, glutamic acid, serine and glycine. The molecular weight calculated from the total amino acid residues was found to be 17,000.Alanine is the NH2 terminal amino acid.The peptide maps obtained after degrading ATPase I with cyanogen bromide or trypsin are in accordance with the methionine, lysine and arginine residues we found in the ATPase I molecule.ATPase I does not appear to be a glycoprotein.     

Reactive Site of API-2c,an Alkaline Protease Inhibitor,for Subtilisin BPN′

Differences between α- and β-lipovitellin were examined, especially in regard to the polypeptide and carbohydrate composition of apolipoprotein. Both lipoproteins were composed of at least eight polypeptides with similar molecular weights ranging from 35,000 to 140,000 daltons. Polypeptides with 110,000 daltons were common major constituents. The close similarity of component polypeptides in the two lipoproteins was also assumed from similar amino acid compositions and the identical immunological properties of the two lipoproteins. However, some notable differences were found in the composition of the polypeptides. α-Lipovitellin contained much more polypeptide with 85,000 daltons than β-lipovitellin. Both apolipovitellins were found to be glycoprotein containing mannose, galactose, glucosamine and sialic acid. The sialic acid in α-lipovitellin exceeded that in β-lipovitellin by six times, though only slight differences were found in the content of neutral and amino sugars. The relatively acidic nature of a-lipovitellin compared with β-lipovitellin is attributed not only to the relative predominance in protein phosphorus but also to the predominance in the sialic acid.     

Linker histone H1.b is polymorphic in grey partridge (Perdix perdix)

This study was aimed at characterizing allelic variations of erythrocyte histone H1.b by comparing the electrophoretic patterns of histone H1.b from individuals of grey partridge (Perdix perdix) population. As two alloforms, H1.b1 and H1.b2, were discerned in the screening gels, the histone H1.b was regarded to be a polymorphic protein encoded by a gene with two codominant alleles, b1 and b2, at a locus. The tested population was found to be at Hardy-Weinberg equilibrium (chi2 = 0.834, p = 0.361), with only a minor heterozygote deficiency (fixation index F = 0.136). Since the histone H1.b alloforms were identified in a two-dimensional gel containing sodium dodecyl sulfate, with no significant differences in their migration pattern in an one-dimensional acetic acid polyacrylamide gel, we assumed that the H1.b alloforms possessed a similar net charge and differed in their apparent molecular weights. A comparison of N-bromosuccinimide-cleaved and alpha-chymotrypsin-digested products of histone H1.b alloforms revealed slight differences in the velocity of C-terminal peptides and a similarity in migration of their N-terminal peptides in one-dimensional sodium dodecyl sulfate-polyacrylamide gel. Therefore, it seemed that the histone H1.b alloforms might differ in this amino acid sequence in a protein segment between N-bromosuccinimide cleavage site and the very C-terminus.     

Structural studies on individual components of bovine transferrin

The single-banding components of bovine transferrin from animals homozygous for the four transferrin variants found in the U.K. were isolated. Sedimentation equilibrium ultracentrifugation and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis showed that the bands of a single variant have molecular weights of 77500 and 73300 respectively. The different bands of a single variant and single bands of different variants show no evidence of size heterogeneity or of low-molecular-weight peptides being split off after reduction in 6m-guanidine hydrochloride. The two slower bands of a single variant, which both contain 2 molecules of sialic acid/molecule of protein, have the same molecular weight and amino acid composition, and give identical peptide ;maps', although differences in composition and peptide ;maps' occur between the different variants. The results support the concept that bovine transferrin is essentially a single polypeptide chain, but they do not explain differences in electrophoretic mobility between bands of the same variant which are not produced by differing sialic acid content.     

Purification and characterization of plasma T-kininogen from Wistar and brown Norway rats

A rapid and convenient three-step purification scheme has been developed for the purification of T-kininogen (alpha 1-cysteine proteinase inhibitor) from rat plasma. The purification process includes chromatography on hydroxyapatite, immunoaffinity chromatography and gel filtration. This procedure is applied to plasma from the brown Norway rat which is known to be deficient in high and low molecular weight kininogens. The method furnished large amounts of T-kininogen from turpentine-treated Wistar rats as well as from untreated and turpentine-treated deficient brown Norway rats. The amino acid and hexose content of the three T-kininogens has been determined. While the composition of the molecules isolated from both injured rats was similar, the neutral sugar content of T-kininogen purified from untreated brown Norway rats was lower and its amino acid composition showed slight differences. The three molecules have identical behaviour and similar physicochemical and immunological properties when analysed by SDS electrophoresis, isoelectrofocusing and two-dimensional immunoelectrophoresis.     

Synthesis of Aspartyl Tripeptide Esters in Relation to Structural Features of Sweet Peptides

The distribution of multiple forms of β-amylase in some varieties or species of soybean seeds was examined by the gel isoelectric focusing method. Seven components (1′, 1, 2, 3, 4, 5 and 6) were found. Their respective isoelectric points were 5.07, 5.15, 5.25, 5.40, 5.55, 5.70 and 5.93±0.04. The varieties or species of soybean seeds were separated into two types by their zymograph: the low pI type and high pI type. Component 6 was purified from commercial defatted soybean meal containing all seven components by ion-exchange column chromatography and by gel filtration, and compared with previously purified components 2 and 4. Components 2, 4 and 6 had the same molecular weight and immunological properties but some differences were found in their amino acid compositions and enzymatic properties. The C-terminal amino acid of components 2 and 6 was glycine but that of component 4 was alanine. It was concluded from these results that differences between components 2, 4 and 6 were caused by charged amino acid substitution.     

Preparation and characterization of troponin C from bullfrog skeletal muscle.

Troponin C was isolated from the skeletal muscle of bullfrog (Rana catesbeiana), and its relative molecular mass was estimated to be 18,000 by SDS/polyacrylamide gel electrophoresis. In its amino acid composition, bullfrog troponin C was similar to that of the frog (Rana esculenta) but different from that of rabbit. Its ultraviolet spectrum was consistent with its amino acid composition. The ultraviolet difference spectrum of the Ca(2+)-loaded form vs. the metal-free form indicated that the single Tyr residue and some Phe residues in the bullfrog troponin C molecule were affected by the conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the metal-free and Mg(2+)-loaded forms migrated slower than the Ca(2+)-loaded form. The property is shared by rabbit troponin C but not parvalbumins or calmodulin. The ATPase activity of CDTA-treated myofibrils reconstituted with bullfrog troponin C showed the same Ca(2+)- and Sr(2+)-sensitivity as that of those reconstituted with rabbit troponin C. Bullfrog troponin C is, thus, physiologically the same as rabbit troponin C, in spite of several marked differences in their physicochemical properties.     

Evolutionary changes reflected by the cellular amino acid composition

Summary Comparison of the amino acid composition of cell-proteins using 17 amino acids has been used to investigate the biological evolution of organisms such as bacteria, blue-green alga, green alga, fungi, slime mold, protozoa and vertebrates. The degree of difference in the amino acid ratios between any two groups reflects the degree of divergency in biological evolution. The amino acid composition of the Gram-negative bacteria (Escherichia coli,Klebsiella,Proteus, andVibrio alginolyticus) was identical. However, the amino acid composition ofStaphylococcus aureus andBacillus subtilis, which are Gram-positive bacteria, differed from each other and from the Gram-negative bacteria. The amino acid composition of the blue-green alga (Cyanobacterium,Chroococidiopsis) was quite similar to that ofE. coli. A marked difference in the amino acid composition was observed betweenE. coli and green alga (Chlorella), and significant differences were observed betweenE. coli and other organisms, such as fungi, protozoa (Tetrahymena), slime mold (Dictyostelium discoideum) and vertebrates. In conclusion, the change in cellular amino acid composition reflects the divergence which has occurred during biological evolution, whereas a basic pattern of amino acid composition is maintained in spite of a long period of evolutional divergence among the various organisms. Thus, it is proposed that the primitive life forms established at the end of prebiotic evolution had a similar amino acid composition.     

Characterization of major proteins in sweet potato tuberous roots

The tuberous roots, but not other organs, of sweet potato contained large quantities of two proteins which accounted for more than 80% of the total proteins. The two proteins, tentatively named sporamins A and B, were monomeric forms with similar M,s (25 000). They were separated from each other by electrophoresis on polyacrylamide gels in a non-denaturing buffer or a buffer containing sodium dodecyl sulphate without being reduced by dithiothreitol. They were very similar to each other with respect to amino acid composition, peptide map and immunological properties. These proteins decreased in preference to other proteins during sprouting. The amino acid sequencing of the amino terminal part of sporamin A suggested that it consists of at least two molecular species with different combinations of a few amino acids.     

Conservation of the basic pattern of cellular amino acid composition of archaeobacteria during biological evolution and the putative amino acid composition of primitive life forms

Summary. Previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs such as eubacteria, protozoa, fungi and mammalian cells. These results suggest that the difference in the cellular amino acid composition reflects biological changes as the result of evolution. However, the basic pattern of cellular amino acid composition was relatively constant in all organisms examined. In the present study, we examined archaeobacteria, because they are considered important in understanding the relationship between biological evolution and cellular amino acid composition. The cellular amino acid compositions of Archaeoglobus fulgidus, Pyrococcus horikoshii, Methanobacterium thermoautotrophicum and Methanococcus jannaschii differed slightly from each other, but were similar to those determined from codon usage data, based on the complete genomes. Thus, the cellular amino acid composition reflects biological evolution. We suggest that primitive forms of life appearing on earth at the end of prebiotic evolution had a similar-cellular amino acid composition. Received November 28, 2000 Accepted January 30, 2001     

Modification of recombinant maize ChitA chitinase by fungal chitinase-modifying proteins

In commercial maize, there are at least two different alleles of the chiA gene that encode alloforms of ChitA chitinase, a protein that is abundant in developing seed. Both known alloforms are modified by Bz-cmp, a chitinase-modifying protein (cmp) secreted by the fungal pathogen Bipolaris zeicola. One alloform (ChitA-B73) is also modified by Stm-cmp, a protein secreted by the fungal pathogen Stenocarpella maydis, whereas the other (ChitA-LH82) is resistant. The two ChitA alloforms possess six differences or polymorphisms (P1-P6). To determine whether the P2 polymorphism in the chitin-binding domain is responsible for resistance or susceptibility to modification by Stm-cmp, and to determine whether Stm-cmp and Bz-cmp are proteases, heterologous expression strains of the yeast Pichia pastoris that produce recombinant maize ChitA (rChitA) alloforms and mutant rChitAs were created. rChitA alloforms and mutant rChitAs were purified from yeast cultures and used as substrates in assays with Stm-cmp and Bz-cmp. As with native protein, Bz-cmp modified both rChitA-LH82 and rChitA-B73, whereas Stm-cmp modified rChitA-B73 only. Mutant rChitAs, in which the P2 amino acids were changed to those of the other alloform, resulted in a significant exchange in Stm-cmp susceptibility. Amino-terminal sequencing of unmodified and modified rChitA-B73 demonstrated that Stm-cmp cleaves the peptide bond on the amino-terminal side of the P2 alanine, whereas Bz-cmp cleaves in the poly-glycine hinge region, the site of P3. The results demonstrate that Stm-cmp and Bz-cmp are proteases that truncate ChitA chitinase at the amino terminus, but at different sites. Both sites correspond to polymorphisms in the two alloforms, suggesting that the sequence diversity at P2 and P3 is the result of selective pressure to prevent truncation by fungal proteases.     

The amino acid composition of mammalian and bacterial cells

Summary High performance liquid chromatography was used to analyze the amino acid composition of cells. A total of 17 amino acids was analyzed. This method was used to compare the amino acid compositions of the following combinations: primary culture and established cells, normal and transformed cells, mammalian and bacterial cells, andEscherichia coli andStaphylococcus aureus. The amino acid compositions of mammalian cells were similar, but the amino acid compositions ofEscherichia coli andStaphylococcus aureus differed not only from mammalian cells, but also from each other. It was concluded that amino acid composition is almost independent of cell establishment and cell transformation, and that the amino acid compositions of mammalian and bacterial cells differ. Thus, it is likely that changes in amino acid composition due to cell transformation or species differences between mammalian cells are negligible compared with the differences between mammalian and bacterial cells, which are more distantly related.     

On the identification of the uncoupler binding protein of bovine mitochondrial oligomycin sensitive ATPase

The 55,000 dalton polypeptide component of the membrane sector of the mitochondrial oligomycin sensitive ATPase has been purified by recycling chromatography on BioGel P-100. The amino acid composition of the purified polypeptide differs significantly from that of the α-subunit of F₁ with which it shares a similar apparent molecular weight. However, the amino acid composition of the former is identical to that of the Factor B polypeptide, which is known to occur in oligomeric forms. Evidence is presented which suggests that the mitochondrial uncoupler binding proteins and the various oligomeric forms of the Factor B polypeptide, including the 55,000 dalton species described in the present report, are identical.     

Chromatographic identification of a new subform of carbonic anhydrase in rabbit erythrocytes

In addition to the two forms of carbonic anhydrase (CA) known to occur in rabbit erythrocytes, a third enzyme species has been isolated and partially characterized. The three Chromatographic forms elute from diethylaminoethyl-Sephadex roughly in the proportions of 3:5:2, in that order. They do not appear to be interconvertible. Their molecular weights are approximately 30,000. Initial kinetic studies of ester hydrolysis and of CO₂ hydration by these carbonic anhydrases identify the first peak to elute as a low-activity form (CA I) and the following two as high-activity forms (CA II, CA II′). Inhibition studies indicate that equal concentrations of all three enzyme species are equally inhibited by equal concentrations of benzenesulfonamide or acetazolamide. The amino acid compositions of the three species support their identification as high- and low-activity forms and indicate substantial differences between CA I and CA II or CA II′, while the latter two differ only slightly. Peptide maps obtained from tryptic digests show considerable differences between the peptides of the high- and low-activity forms, while the peptides of CA II and CA II′ are almost indistinguishable. The available evidence suggests that the newly isolated CA II′ species is a subform of the major high-activity isoenzyme and that a very slight difference in amino acid composition is responsible for a net charge difference between CA II and CA II′, which is reflected in their different Chromatographic properties.     

Purification,characterization, and synthesis of vitellin from the cabbage butterfly Pieris rapae L.

Vitellin from the cabbage butterfly Pieris rapae L. was purified and characterized by electrophoresis. Vitellin from P. rapae is a phosphorylated glycolipoprotein of 380,000 ± 10,000 molecular weight as determined by nondenaturing polyacrylamide gel electrophoresis. Two subunits with an M_r of 150,000 and 40,000 were obtained from vitellin. The native molecule is thought to be a tetramer composed of two molecules of each of these subunits. The isoelectric point, as determined by isoelectric focusing on polyacrylamide gels, is 6.10. Vitellin and vitellogenin were indistinguishable by immunological methods such as double diffusion and tandem-crossed immunoelectrophoresis. Vitellogenin from the hemolymph and vitellin from the ovary were quantified by rocket immunoelectrophoresis. Vitellogenin and vitellin were first detected in 6-day-old pupae, and their levels increased continuously during ovarian development. Vitellogenin synthesis by the fat body in 4-day-old female pupae could be induced by juvenile hormone I.     

Two isoforms of Rubisco activase in cotton,the products of separate genes not alternative splicing

In several plant species, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase consists of two isoforms that are produced by alternative splicing of a pre-mRNA. Two forms of activase corresponding to the longer, redox-regulated alpha and the shorter, beta forms were detected immunologically in cotton (Gossypium hirsutum L.) leaves, but their N-termini differed in 4 of 14 residues. The cDNAs for the alpha and beta forms of cotton activase diverged throughout the translated and 3'-untranslated regions, including variations that accounted for the differences in N-terminal amino acid sequence. Analysis of genomic DNA confirmed that separate genes encoded the alpha and beta forms of cotton activase. Separate activase genes were also detected in diploid species of cotton containing the different progenitor genomes of the cultivated allotetraploid, indicating that the occurrence of separate alpha- and beta-form genes in cotton predates the merger of the diploid genomes. The deduced amino acid sequences of the two forms of cotton activase exhibited 84% identity and both forms were active after expression in Escherichia coli. The recombinant alpha and beta forms exhibited similar affinities for ATP and only minor differences in thermotolerance, but their ATPase specific activities differed. The results show for the first time a plant species with two forms of activase that are structurally and functionally equivalent to the alternatively spliced alpha and beta forms in other plants, but that are encoded by separate genes. That cotton still expresses both forms of activase, even without alternative splicing, suggests that each form has a required function in photosynthesis.     

Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Biochemical and immunological properties of different electrophoretic forms of juvenile hormone esterase from Trichoplusia ni (Hübner)

The two major electrophoretic forms (pI 5.5, 5.3) of juvenile hormone esterase were independently isolated from hemolymph of larval Trichoplusia ni. A simple and rapid preparation procedure of poly(ethylene glycol) precipitation, Sephadex gel filtration and chromatofocusing is described. Analytical isoelectric focusing showed only one peak of juvenile hormone esterase activity in the respective purified samples, whereas there were four (two major) such peaks in the hemolymph. The amino acid composition of the two forms was similar. The comparison of peptides obtained after protein fragmentation by cyanogen bromide showed that juvenile hormone esterases A and B were very similar, although definitely not identical, in amino acid sequence. The immunological comparisons of juvenile hormone esterases suggested that the number of polyclonal antibody binding sites on both forms was the same. There were no detected differences between immunoreactive properties of juvenile hormone esterase from the hemolymph of different stages of larval maturation. The influence of the active site of the enzyme on its antigenic properties was studied by immunocompetition. The inactive, heat-denatured juvenile hormone esterase can only partially protect against inhibition of its activity by the antibodies, whereas an organophosphate inhibitor which covalently binds to the catalytic center of the enzyme did not change the immunoreactive properties in comparison to active juvenile hormone esterase from hemolymph. These data show that heat-denatured juvenile hormone esterase has lost at least one or more epitopes, but the catalytic site of the enzyme is distinct from the epitopes.     

Studies of soluble rat liver mitochondrial acid ATPases. II. Structural and immunological properties of ATPase 1.

We described previously the existence of a soluble ATPase activity in rat liver mitochondria [1]. The purification and catalytic properties have been described [2]. In a continuation of these experiments, we have studied the immunologic and structural properties of one molecular form of this enzyme : ATPase I. We have prepared the antiserum anti-ATPase I and demonstrated the purity of our enzyme preparation by immunodiffusion and immunoelectrophoresis. An immunohistochemical method also confirmed the localization of ATPase I in the soluble fraction of mitochondria. The molecular weight of ATPase I was measured by G 100 Sephadex gel filtration and was found to be 18,400; electrophoresis on polyacrylamide gels gave a value of 18,600. The pHi of ATPase I was found to be 7,2. Amino acid analysis showed high amounts of aspartic acid, glutamic acid, serine and glycine. The molecular weight calculated from the total amino acid residues was found to be 17,000. Alanine is the NH2 terminal amino acid. The peptide maps obtained after degrading ATPase I with cyanogen bromide or trypsin are in accordance with the methionine, lysine and arginine residues we found in the ATPase I molecule. ATPase I does not appear to be a glycoprotein.     

Amino acid composition and immunochemical properties of AcPase III and AcPase IV representing glycoforms of the lower molecular weight, tartrate-resistant acid phosphatase of the frog liver.

1. Amino acid composition and immunological properties of the frog liver LMW AcPase forms: AcPase III and IV were examined. 2. AcPase III and IV show nearly identical amino acid composition and close immunological similarity. 3. These results indicate protein identity of both the enzyme forms and together with our previous data [Kubicz A., Szalewicz A. and Chrambach A., Int. J. Biochem. 23, 413-419 (1991)] demonstrate that generation of AcPase III and IV is a modification of the same enzyme protein by glycosylation processes. 4. Differences in immunoreactivity between AcPase III and IV were observed and discussed to be due to their altered conformations.