Differences in cattle globins

1. Comparisons have been made between electrophoretic mobilities of the cattle haemoglobins, HbA, HbB, HbC, HbD and HbF, at pH8.9. The fastest was HbB, then came in decreasing order HbF, HbC, HbA and HbD. 2. Globins were prepared from the main fractions of the five haemoglobins by CM-cellulose chromatography and investigated by starch-gel electrophoresis in four different buffer systems. In three of these (pH2.0 and 11.8) the globins appeared as two bands on stained starch gels. The slowest bands, the alpha-chains, showed the same rate of migration in all five globins. The faster bands, the non-alpha-chains, differed, that of HbF being the fastest and that from HbC the slowest. The other three were intermediate with, however, very small difference between the non-alpha-chains from HbA and HbD. 3. At pH1.8 in an acetate-phosphate-hydrochloric acid-urea buffer three bands appeared in all five globins of which the two slowest were indistinguishable in rates of migration, whereas the rates of migration of the third and fastest bands differed. Explanations for the occurrence of three bands are discussed.     

Electrophoretic study of myosin isoforms in white muscles of some teleost fishes

1. White skeletal muscle myosin of four marine teleost fish species (cod, blue whiting, Norway haddock, and spotted wolf-fish) was analyzed by native, SDS-PAGE, and 2-dimensional electrophoresis. 2. Four types of native myosin were present in cod, blue whiting and Norway haddock. The second fastest migrating form was predominant. 3. Myosin from spotted wolf-fish also resolved into four forms. The fastest migrating form was hardly noticeable. The other three were present in apparently similar amounts. 4. In the myosin from each species there were three types of light chains. The pattern of light chains was species specific. 5. Apparently, there was only one type of heavy chain in myosin from cod, Norway haddock and spotted wolf-fish. One preparation of cod showed an extra band of higher electrophoretic mobility than the main band. In blue whiting we found two bands present in approximately equal amounts.     

Foetal and neonatal transferrins in cattle

Homozygous transferrins of adult cattle are made up of two strong pairs and one weak pair of protein bands on starch gel electrophoresis. Foetal transferrins have only the slower band of each pair with the fastest band of the three being much stronger than in the adult type. Before term the second band of each pair begins to develop and at the same time the fastest pair becomes weaker – attaining the adult type by term or soon after. The ai protein, which is present in early foetal life and almost disappears by 250 days of embryonic development, shows individual variation. Its relationship to fetuin is discussed.     

Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111

Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR. All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone. The remaining fraction was shown to be phosphatidylethanolamine. The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents. The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions. The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups. Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions. In summary, the heterogeneity of E. coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.     

Changes of aspartate aminotransferase activity, its isoform pattern, and free amino acids content in peanut cotyledons during seed germination

After imbibition of peanut seeds, the concentration of free amino acids in cotyledons increases probably due to hydrolysis of reserve proteins. That increase was accompanied by a stimulation of aspartate aminotransferase (AAT, EC 2.6.1.1) activity especially pronounced between first and sixth day of imbibition. Peanut cotyledons contained several AAT isoforms which could be visualized after polyacrylamide gel electrophoresis (PAGE). Native PAGE of extracts from cotyledons of dry seeds demonstrate three active bands. The intensity of these bands increased reaching a maximum after 6th day of imbibition. An additional band appears transientely on 6th day, but is not visible after 14 days of incubation. A fastest band appears on this latter day. If germination takes place under hypersalinity conditions. the AAT activity severely diminishes and the free amino acid concentration raises. The possibilities of different types of AAT regulation are discussed.     

Interpretation of triose phosphate isomerase isozymes in the cherimoya (Annona cherimola Mill.)

Detailed interpretation of triose phosphate isomerase (TPI) isozymes in seed plants has been restricted to only a few species. Three sets of TPI bands are regularly observed in the cherimoya(Annona cherimola), a primitive angiosperm. The slowest, set I, is expressed as one or three bands; the second-slowest set II, as one or two bands; and the fastest, set IV, as one or three bands. A faint set III, just cathodal to set IV, is detected rarely with overstaining. Set IV bands are expressed in macerated extracted pollen but not in pollen leachate. Dissociation-reassociation experiments reveal that the set II bands are heterodimers involving, in part, the enzymes involved in the set I bands. These data combined with those from full-sib progeny analysis lead us to propose a three-locus model to explain the TPI isozyme banding patterns in cherimoya. Sets I and IV consist of the allelic products of individual, single loci. Sets I and II occur in the cytoplasm. Set IV occurs in organelles. Set II isozymes are the intergenic heterodimers of the locus coding for set I and the locus coding for set III. Our results reported here are contrasted with the TPI isozyme patterns known for other vascular plants and suggest that the locus coding for set III may be a duplication of very ancient origin.This work was supported, in part, by funding from the Elvenia J. Slosson Endowment Fund.     

Analysis of the multiple forms of Gaucher spleen sphingolipid activator protein 2.

Gaucher spleen sphingolipid activator protein 2 was fractionated into concanavalin A binding- and non-binding fractions. These fractions each contained several bands on non-denaturing polyacrylamide gel electrophoresis (PAGE). The two fractions were further fractionated by electroblotting the proteins from preparative gels onto nitrocellulose, staining with Ponceau S to locate the bands of protein and then eluting the protein components from the nitrocellulose. A total of ten fractions, each containing only one or two major components, was collected. All of these subfractions activated beta-glucocerebrosidase and sphingomyelinase and most subfractions also activated beta-galactocerebrosidase. The structural relationship of the bands was investigated using endoglycosidase digestions. The results indicated that the two bands with the fastest mobility on non-denaturing PAGE did not contain any carbohydrate. The remaining bands showed only limited or partial digestion with endoglycosidase H and endoglycosidase D, but were readily hydrolysed with endoglycosidase F. The products of these digestions included bands with similar mobilities to the non-carbohydrate containing bands.     

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.     

Myosin heavy-chain-based isomyosins in developing, adult fast-twitch and slow-twitch muscles.

A modified method of electrophoresis under nondenaturing conditions made it possible to separate rat muscle extracts of defined myosin heavy chain (HC) and light chain (LC) composition into subsets of developmental, fast and slow myosin heavy-chain-based isomyosins. The fastest migrating isomyosins were the neonatal isomyosins (nM1, nM2, nM3), followed by the slightly slower migrating embryonic isomyosins (eM1, eM2, eM3, eM4). Of the nine adult fast isomyosins, the HCIIb-based isomyosins (FM1b, FM2b, FM3b) were the fastest migrating. These were followed by the HCIId-based isomyosins (FM1d, FM2d, FM3d). The HCIIa-based isomyosins (FM1a, FM2a, FM3a) were the slowest. Our results suggest that FM3a is identical with the so-called intermediate isomyosin (IM) described in the literature. The slow myosin heavy-chain-based isomyosins (SM1, SM2, SM3) migrated far behind the fast isomyosins. Whereas the gross electrophoretic mobilities of each of these isomyosin triplets is determined by the specific heavy chain complement, the different mobilities of the bands within each triplet result from different alkali light chain combinations. Thus, the fastest triplet bands of the neonatal (nM1) and adult fast isomyosins (FM1b, FM1d, FM1a) represent the LC3f homodimers, the slowest (nM3, FM3b, FM3d, FM3a) the LC1f homodimers, and the intermediate bands (nM2, FM2b, FM2d, FM2a) the LC1f/LC3f heterodimers. Different proportions of the adult fast isomyosin triplet bands indicate that the affinity for LC3f decreases in the order HCIIb, HCIId, HCIIa. The three slow isomyosins represent LC1sa (SM1) and LC1sb (SM3) homodimers and a LC1sa/LC1sb heterodimer (SM2). Circumstantial evidence suggests an inverse order in rabbit muscle where SM1 and SM3 most likely represent LC1sb and LC1sa homodimers, respectively.     

An electrophoretic study of the low-molecular-weight components of myosin

1. The low-molecular-weight components of myosin freshly prepared by the standard procedure from adult rabbit skeletal muscle migrated as four main bands Ml(1), Ml(2), Ml(3) and Ml(4) on polyacrylamide-gel electrophoresis in 8m-urea. 2. The number of bands increased on storage. This change was accelerated by increasing the temperature and pH. 3. None of the bands had electrophoretic mobilities identical with those of the well-characterized proteins of the myofibril or with the sarcoplasmic proteins. 4. By varying the ionic conditions and concentration of muscle mince used for the initial extraction it was possible to change the relative proportions of the two electrophoretic bands of intermediate mobility, Ml(2) and Ml(3). 5. The four-band picture similar to that obtained with rabbit was observed with myosin isolated from skeletal muscle of the rat, mouse, hamster, pigeon and chicken. 6. Rabbit cardiac myosin gave only two bands on electrophoresis. Myosin from rabbit red muscle gave a pattern intermediate between cardiac and white-skeletal-muscle myosin, i.e. the two fastest bands were present in decreased relative amounts. 7. It is suggested that the differences in the low-molecular-weight components of myosin from different types of muscle are a consequence of differences in the isoenzyme composition of the myosins.     

Individual expression patterns of myotoxin isoforms in the venom of the snake Bothrops asper.

1. In order to investigate the distribution of myotoxin isoforms in the snake Bothrops asper, venoms from individual specimens were analyzed by a cathodic electrophoretic system for basic proteins under native conditions. 2. The electrophoretic system resolved at least five bands with slight differences in mobility, corresponding to the fastest migrating proteins in the venom. The identity of the bands was confirmed by immunoblotting, using a rabbit anti-myotoxin serum. 3. There were clear differences in the individual patterns of myotoxin isoform expression, both in specimens from the Atlantic and Pacific regions of Costa Rica. Some individuals possessed all five variants. 4. In agreement with previous reports, the venoms of ten newborn (less than 10 days of age) specimens completely lacked myotoxin bands, indicating an ontogenetic regulation in the expression of these toxins in B. asper. 5. One of the bands, corresponding to the lysine-49 phospholipase myotoxin II, was the only isoform present in all individuals studied, suggesting a possible selective pressure for the conservation of this type of protein in the venom of B. asper.     

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.     

Alkaline Band Formation in Chara corallina: Due to OH? Efflux or H+ Influx?

The nature of the transport system responsible for the establishment of alkaline bands on cells of Chara corallina was investigated. The transport process was found to be insensitive to external pH, provided the value was above a certain threshold. At this threshold (pH 5.1 to 4.8) the transport process was inactivated. Transport function could be recovered by raising the pH value of the external solution. The fastest rate of recovery was always obtained in the presence of exogenous HCO₃⁻.     

Inheritance of electrophoretic variants of tuber proteins in Solanum tuberosum haploids

Soluble tuber proteins were separated by discontinuous polyacrylamide gel electrophoresis on vertical slabs. Banding patterns of proteins stained with Coomassie Blue in 7.5% acrylamide gels (pH 4.3) were few and distinctive for haploids (2n = 2x = 24) derived from several cultivars (2n = 4x = 48). Katahdin and Chippewa haploids have only three different banding patterns for the eight fastest moving bands. The haploids have either the parental pattern (all eight bands) or one of two complementary banding patterns (four bands). The frequency of these patterns among the haploids indicates that the eight bands are controlled by one locus which is duplex (A¹A¹A²A²) in the parents. Haploids with the genotype A¹A² have eight bands. A¹A¹ haploids have four bands, and A²A² have the other four bands. Tawa haploids have in equal numbers either the eight (A¹A²) or four (A²A²) band pattern. Thus the genotype of Tawa is A¹A²A²A². The control of four bands by one allele could be explained by assuming that these alleles are involved in posttranslational modification or assembly of one or two protein species. Another explanation is that pseudoalleles or redundant genes produce the groups of protein bands. The eight proteins studied apparently are of similar molecular weight but differ in charge.     

The laminar organization of optic nerve fibres in the tectum of goldfish.

Potentials in the tectum of large (12--20 cm) goldfish, evoked by stimulation of the optic nerve, were recorded extracellularly with double-barrelled electrodes (d.c., saline and a.c., Woods metal--Pt). Four fibre groups (E, M1, M2, M3) were recorded at latencies of approximately 2, 3, 5 and 8 ms after stimulation (conduction velocities of approximately 7, 5, 3 and 2 m/s). The same four groups were recorded from the optic nerve when the tectum was stimulated. The fastest fibre groups (E) did not give rise to a postsynaptic wave. Fibre groups M1, M2 and M3 gave rise to postsynaptic potentials which, following computation of their second spatial derivatives with depth, were found to have current sinks at depths of approximately 100-150 micrometers, 150--200 micrometers and 250--350 micrometers respectively. Thus the fastest conducting retinotectal fibres make their synapses most superficially, the opposite of the arrangement in the frog tectum. These postsynaptic waves fatigued at repetitive stimulus rates of 20--50 per second, and in twin pulses at interstimulus intervals of 10--15 ms; and they were reversibly blocked by topical application of pentobarbitol. The fibre potentials, however, were virtually undecremented under these conditions. To compare these electrophysiological findings with the anatomy, the cobalt procedure was used to visualize the profiles of the optic fibres in the various tectal laminae. A thick dense projection filled the superficial grey and white (s.g.w.) layer, and there was a thin satellite band just superficial to it. In addition, there were two deeper bands of sparse innervation, in the middle of the central grey zone (c.g.) and in the deep white (d.w.) layer. These bands were associated with the field potential sinks through lesions made with recording electrodes. The two deep bands correspond to the M3 fibre group. The dense s.g.w. innervation contains both the M1 and M2 fibre groups, the M1 just superficial to the M2. The fastest fibre group, E, which had no postsynaptic wave associated with it, persisted at least six weeks after retinal removal, and probably represents efferent cells with fibres projecting back through the optic nerve to the retina. Filled cell profiles could not be positively identified with the cobalt technique, but could be seen with the HRP technique, when the optic afferents were first allowed to degenerate. The filled cells were the pyramidals of the s.g.w. layer.     

Properties and Subunit Characterization of Affinity Purified Sophora Japonica Lectin

The affinity purified Sophora japoniea lectin exhibits an anomalous behavior on polyacrylamide gel electrophoresis (PAGE). Electrophoresis at pH 8.9 produces three protein staining bands. Extraction and re-electrophoresis of the fastest and slowest migrating components demonstrates that the lectin solution is an equilibrium mixture of interconvertible forms. Addition of a bindable saccharide, D-galactose, during PAGE causes the equilibrium to be shifted toward a single form. As indicated by analytical gel filtration, sedimentation velocity ultracentrifugation and ion-exchange chromatography experiments, the equilibrium mixture consists of charge and not molecular weight variants of the native molecule of 132,800 g/m. Results from end-group and cysteine analyses and PAGE in sodium dodecyl sulfate indicate that the native lectin is composed of the non-covalent association of two dissimilar subunits. One subunit consists of two identical polypeptide chains attached by two disulfide bonds and the other subunit of two identical polypeptide chains stabilized by a single cysteine bridge.     

Migration patterns of proteins in ancient human bones detected by SDS electrophoresis

Purified and ultraconcentrated extracts of 7 burial remains from different areas and various periods (from IV B.C. to 1300–1400 A.D.) were repeatedly submitted to electrophoresis in SDS. Five bands: A, B, C, D, E (from the slowest to the fastest) were identified altogether. Band B appears to be the most resistant, because it is present in all the samples with reactivity. Proteins corresponding to the five bands have molecular weights comprised between 65000 and 45000 in agreement with other authors.     

One-lane sequence analysis of oligodeoxyribonucleotides

Treatment of 5'-end 32P-labeled oligodeoxyribonucleotides with 0.4 M aqueous piperidinium formate, pH 2, at 37 degrees C for 6 h, followed by treatment with 1 M aqueous piperidine at 90 degrees C for 6 h, produces, after electrophoresis through 27% polyacrylamide sequencing gels, one-dimensional distributions of radioactivity from which the base sequences can be deduced. The order of intensities for the bands signaling the various bases is G greater than A greater than C greater than T. The spacing from a given band to the next higher band in the ladder was base characteristic, the order of band spacings being G greater than T greater than or equal to A greater than C. In contrast to the one-cleavage one-lane DNA sequencing method reported earlier (B. J. B. Ambrose and R. C. Pless, 1985, Biochemistry 24, 6194-6200), which was based on treatment of end-labeled DNA with hot aqueous piperidine in the presence of sodium chloride, the present method produces a salt-free hydrolysate, thus minimizing electrophoretic irregularities in the fastest moving bands.     

Genetic variation in supernatant malate dehydrogenase of birds and reptiles

Electrophoretic variants of supernatant malate dehydrogenase were found within populations of a brid (Pitohui kirhocephalus) and of a reptile (Chrysemys picta). Upon starch gel electrophoresis, tissue extracts from variant individuals exhibit three equally spaced bands of supernatant malate dehydrogenase activity, whose staining intensities are approximately in a ratio of 1:2:1. Normal individuals, on the other hand, exhibit a single major band which is identical in mobility with the fastest of the variant bands. Immunological tests show that the Pitohui and Chrysemys enzymes are homologous with the well-characterized supernatant malate dehydrogenase of the domestic chicken. These results along with those obtained on other species imply that birds, reptiles, mammals, and certain invertebrates may possess a single locus coding for supernatant malate dehydrogenase. By contrast, some species of fishes and invertebrates possess multiple loci for this enzyme. A notable feature of the present investigation was the use of phenoxyethanol for extracting malate dehydrogenases from bird tissues. Extracts were prepared simply by soaking tissue in an aqueous solution of this reagent, no homogenization or centrifugation being required.This investigation was supported by funds from a research grant awarded by the National Science Foundation (GB-13119) and a U.S. Public Health Service Training Grant (GM 31-12).     

穴居狼蛛毒中一个抗菌活性多肽的鉴定和纯化

从我国新疆地区产穴居狼蛛的毒液中分离纯化了一种多肽——狼蛛抗菌肽(Lycosin)。穴居狼蛛的粗毒在酸性聚丙烯酰胺凝胶电泳中可分出11条蛋白带,用0.6％的大肠杆菌琼脂胶覆盖在凝胶上进行鉴定表明,电泳迁移率最快的区带有抗菌活性。经鉴定该多肽分子系由43至45个氨基酸残基组成,N-末端为丙氨酸,分子中的碱性和疏水性氨基酸分别占总氨基酸残基数的1/4和1/3。