Major Electrophoretic Components Research Articles

Immunological response and ovarian histology of squirrel monkeys (Saimiri sciureus) immunized with porcine zona pellucida ZP3 (Mr = 55,000) macromolecules.

ZP3 (M, = 55,000) is the major electrophoretic component of the porcine zona pellucida (ZP). In a continuing assessment of ZP3 as a candidate antigen for contraceptive vaccine development, female squirrel monkeys were immunized with 200 μg ZP3 using either Freund's adjuvant (FA) or muramyl dipeptide (MDP) and the effect of such immunization on ovarian histology examined. Two experimental and three control groups were immunized: Group 1 (n = 4), ZP3 plus FA; Group 2 (n = 4),ZP3 plus MDP; and controls-Group 3 (n = 2), ZP3 alone; Group 4 (n = 4), FA alone; and Group 5 (n = 4), saline. High antibody response to ZP3 was detected in the ZP3/FA and ZP3/MDP groups, and a very low response, in the ZP3-alone group. Immune profiles for the ZP3iFA and ZP3/MDP groups were comparable, but titers in the MDP group were consistently lower and decreased more rapidly after 300 days post-immunization (PI) than in the FA group. At 6 months PI, all ovaries from the ZP3/FA group revealed a deficiency of zona-encased oocytes and a reduction in secondary and tertiary follicles compared to controls. At 18-24 months PI, normal ovarian histology in one ZP3/FA injected monkey and the presence of zona-encased oocytes in a second monkey suggested ovarian recovery. Normal ovarian histology was present in all monkeys in the ZP3/MDP group as well as in all controls. These findings indicate that while immunization with ZP3/FA does initially perturb normal ovarian histology, such adverse effects appear to be reversible. Furthermore, immunization using ZP3 with MDP has no adverse effect on the ovary, indicating the importance of proper adjuvant selection in immunocontraceptive (IC) studies. These data encourage continued investigation of the zona IC approach using well-characterized zona immunogens with non-Freund's adjuvants.

Functional properties of the two major hemoglobin components from Leporinus friderici (Pisces)

1. 1. The hemoglobins of Leporinus friderici were separated by liquid chromatography on DEAE-Sepharose in order to isolate the two major electrophoretic components. 2. 2. The chromatographic fraction I (electrophoretically slow anodic) showed no Bohr effect and no nucleoside triphosphate modulation. 3. 3. The chromatographic fraction III (electrophoretically fast anodic) showed a normal Bohr effect and addition of nucleoside triphosphate decreased oxygen affinity but did not alter the Bohr effect. 4. 4. The whole hemolysate showed a normal Bohr effect and phosphate modulation altered both Bohr effect and oxygen affinity. 5. 5. No or little difference between the effect of adenosine or guanosine triphosphates on hemoglobin function was observed.

Immunochemical studies on alcoholic hyalin.

Alcoholic hyalin was isolated from postmortem human livers with alcoholic hyalin bodies using discontinuous sucrose density gradient centrifugation and two-phase polymer system. Rabbit gamma globulin antibody to alcoholic hyalin was obtained at 0.18-0.33 ammonium sulfate saturation and made specific by absorption with normal human liver homogenate. The specific antibody was found to react with alcoholic hyalin present in tissue sections of human livers with alcoholic hepatitis and also those present in smears as revealed by immunoperoxidase staining. Gel diffusion analysis with the specific antibody showed precipitin arcs against alcoholic hyalin and also a fraction from normal human liver prepared by an identical procedure for alcoholic hyalin. When this antibody was further absorbed with normal human liver homogenate suspended in 0.01 M Na phosphate buffer pH 7.4: 0.15 M sodium cloride containing 0.1% sodium dodecyl sulfate, precipitin arcs were only observed with alcoholic hyalin. Rabbit gamma globulin antibody to mouse liver cell nucleus reacted with normal human liver cell nucleus and with alcoholic hyalin. Rabbit gamma globulin antibody to mouse liver cell membrane produced precipitin arcs against normal human liver cell membrane and against alcoholic hyalin. Sodium dodecyl sulfate acrylamide gel electrophoresis of alcoholic hyalin revealed the presence of six major electrophoretic components of which approximate molecular weights are from 35,000 to 140,000. One protein component of approximate molecular weight 100,000 showed antigenic reactivity against the antibody specific for alcoholic hyalin, while other electrophoretic components reacted only with unabsorbed antibody preparations. Amino acid composition of the isolated electrophoretic components presented similarities and dissimilarities between these components. The present investigation indicates the presence of normal human liver cell components in alcoholic hyalin and that of limited antigenic determinents of alcoholic hyalin.

Silkmoth chorion proteins: sequence analysis of the products of a multigene family.

Five polypeptide components have been isolated from the eggshell (chorions) of a silkmoth. Two are homogeneous on sodium dodecyl sulfate and isoelectric focusing gels, and three contain predominantly two proteins each. Amino acid analyses show that all five components are similar to each other. These proteins have been sequenced from the amino terminus. Homogeneous components yielded single sequences; heterogeneous components yielded two residues at some positions, consistent with their containing two major electrophoretic components. Striking similarities are apparent among all these sequences. These similarities can be increased dramatically by separating each of the three protein mixtures into two sequences and introducing a small number of gaps or insertions. This is due in part to bringing into register a portion that contains short repeating subunits found in all sequences. All proteins are also characterized by a region of high cysteine content near the amino terminus followed by a longer low-cysteine region. The data suggest that these proteins share a common evolutionary origin and are encoded by a multigene family.

Metabolic properties of the products of mitochondrial protein synthesis in HeLa cells.

The metabolic behavior of the mitochondrial protein synthesis products has been investigated in HeLa cells. Particular attention was given to the four major electrophoretic components (designated as Nos. 2, 3, 5, and 8) of the 10 previously identified as organelle-specific products. Inhibition of cytoplasmic protein synthesis with emetine or cycloheximide causes a rapid decline in the rate of mitochondrial protein synthesis, with an estimated half-life of 1 to 2 h, affecting in a parallel way all the discrete components. About 30% of the original synthetic activity appears to be resistant to emetine treatment for at least 24 h; however, all the polypeptides synthesized after the first 4 h of cell exposure to emetine are metabolically unstable, possibly because of lack of integration into the inner mitochondrial membrane. An analysis of the stability of newly synthesized products of mitochondrial protein synthesis pulse-labeled in the presence of cycloheximide and then chased in the absence of the drug (i.e. under conditions of resumed cytoplasmic protein synthesis) has revealed marked differences among the various discrete components. In particular, about three-fourths of the radioactivity associated with components 3 and 5 decays within 4 h of chase, the remainder being substantailly stable afterwards; by contrast, the radioactivity in components 2 and 8 shows only a slow decline during a 3-day chase. If the chase is carried out under conditions of a persistent block of cytoplasmic protein synthesis, as is the situation after a pulse labeling in the presence of emetine, all newly synthesized components appear to be destablized in various degrees, with the exception of component 5, which is to a great extent stabilized. Inhibition of mitochondrial protein synthesis with chloramphenicol has a progressive stabilizing effect on most of the discrete components newly synthesized after removal of the drug; this effect is especially striking in the case of component 5 which, in experiments of continuous labeling in the presence of emetine after prolonged chloramphenicol treatment, becomes, after 24 h of labeling or more, the only recognizable peak in the electrophoretic pattern of the sodium dodecyl sulfate-lysed mitochondrial fraction. The results of the kinetic experiments described here are interpreted in terms of two roles of cytoplasmically synthesized proteins, one required for the synthesis of polypeptides within the organelles, the other necessary for the stabilization of the mitochondrial products.

The haemoglobin systems of the bloodworms Glycera dibranchiata and G. americana. Oxygen binding properties of haemolysates and component haemoglobins

1. 1. Oxygen equilibria are reported for the haemolysates of Glycera dibranchiata and G. americana and for the polymeric and monomeric fractions and the isolated major electrophoretic components of G. dibranchiata haemoglobin. 2. 2. In contrast to previously published data, no evidence was found for a functionally-significant interaction between the polymeric and monomeric fractions in G. dibranchiata. Two electrophoretic components of the monomer show virtually identical oxygen affinities (P 50, 5 mm at 20°C) and no cooperativity in oxygen-binding. Four major polymeric components show similar oxygen affinities (P 50 ∼ 10–13 mm, at pH 7.3 and 20°C), pH sensitivities (Δ log P 50/Δ pH ∼ 0.3) and cooperativity ( n, 1.2–1.6). 3. 3. G. americana haemolysates reveal oxygen affinities and pH and temperature dependency of oxygen-binding similar to those of G. dibranchiata haemolysates, and a molecular weight heterogeneity indicating the presence of a major tetrameric and a minor dimeric/monomeric haemoglobin fraction. 4. 4. The data are discussed in relation to haemoglobin function in bloodworms.

A Cappelle-Desprez gliadin of high mobility.

AbstractThe fastest major electrophoretic component of the gliadin proteins of Cappelle–Desprez wheat has been purified sufficiently to be characterised. It appears to be a single‐chain, carbohydrate‐free protein, containing no SH groups and having a molecular weight of 37 000. The amino acid composition, with a preponderance of glutamine and proline, is generally typical of the gliadin class. The protein associates appreciably at acid pH.

The Antigens and Autoantigens of the Seminal Vesicle

Abstract Rabbit vesicular fluid (RVF) was shown to contain only one major electrophoretic component. This component, isolated by starch block electrophoresis, was found to have a molecular weight, by determination of sedimentation and diffusion coefficients, of approximately 17,300. This major component was also effective in isoimmunization. Another component, possibly a minor one, had similar antigenic properties. The major component, and the “minor” one also, were found to be highly tissue-specific (that is, accessory glands-specific) and also species-specific. Isoimmunization procedures have led to the production of autoantibodies. No delayed hypersensitivity response or lesions of the accessory glands of reproduction resulted. It was concluded that the major reason no lesions were found was that the rabbit was not able to develop a delayed hypersensitivity type of response to the components of RVF. When rabbit vesicular fluid was ejaculated as part of the seminal plasma, its molecules were still able to induce an autoantibody response by isoimmunization techniques. This was the same as the response induced by rabbit vesicular fluid, itself.

Biological properties of plasmin digests of S-carbamidomethylated human growth hormone.

Reduction and carbamidomethylation of the intrachain disulfide bridges of human growth hormone did not destroy its ability to stimulate weight gain or cartilage metabolism in hypophysectomized rats. The reduced and alkylated hormone also stimulated glucose oxidation in isolated adipose tissue of hypophysectomized rats when added in vitro. When the S-carbamidomethylated hormone was incubated overnight with human plasmin, approximately 95% of the starting material was completely digested, as judged by polyacrylamide gel electrophoresis. The plasmin digest retained the ability to stimulate weight gain, cartilage metabolism, and glucose oxidation. A fraction consisting of two major electrophoretic components was isolated from the digest by chromatography on tsephadex G-50. This fraction possessed the biological properties of the whole digest.

Brine Soluble Protein of Cheddar and Gouda Cheese

Brine soluble proteins of Cheddar and Gouda cheese prepared from milks of different composition were characterized by origin, composition, and electrophoretic properties. The protein originated in the cheese curd, but its composition was not constant throughout aging. Initially, αs1-casein and β-casein were the major electrophoretic components, but 5h after addition of rennet extract two hydrolytic components appeared. In both varieties, the αs1-casein decreased with the production of additional components. Change was less in the β-casein component.Although the electrophoretic patterns of the cheeses were much less distinct than those of the corresponding brine soluble proteins, the number of components was the same in each case.Enrichment of milk with calcium and variety characteristics (i.e., Cheddar or Gouda) had a greater influence on the electrophoretic properties of brine soluble proteins than did the casein/fat ratio of the milk.αs1-casein tended to be hydrolyzed more rapidly in Gouda than in Cheddar cheese.

Characterization of the NH2-terminal Glutamic Acid and NH2-terminal Lysine Forms of Human Plasminogen Isolated by Affinity Chromatography and Isoelectric Focusing Methods

Abstract Human plasminogen was prepared from both plasma Fractions III and III2,3 by affinity chromatography on lysine-substituted Sepharose columns. Plasminogen prepared by this method, from either Fraction III or III2,3, appeared to be homogeneous when analyzed by slab acrylamide gel electrophoresis in 6 m urea-0.1% sodium dodecyl sulfate. The specific activities of the preparations were 20 to 24 casein units per mg of protein. Acrylamide gel electrophoretic analyses in 0.3 m e-aminocaproic acid at pH 8.4 showed that the distribution of the multiple molecular forms of plasminogen prepared from Fraction III differed depending upon whether the extracts were either frozen, or unfrozen, prior to affinity chromatography or processed more quickly during affinity chromatography. The major electrophoretic components in Fraction III plasminogen prepared from extracts which were unfrozen had greater electronegative mobilities than the major components in Fraction III2,3 plasminogen. The electrophoretic forms seen in Fraction III plasminogen prepared from extracts which had been frozen and thawed, prior to affinity chromatography appeared to be identical with those in Fraction III2,3 plasminogen. Electrophoretic analyses in acrylamide gels containing 0.4% casein showed that all of the electrophoretic forms of plasminogen prepared from Fractions III and III2,3 could be activated with urokinase as determined by the zones of casein digestion seen after incubation and staining of the gels. Electrophoretic analyses of Fractions III and III2,3 plasminogens in acrylamide gel-isoelectric focusing systems showed similar patterns to those obtained in the acrylamide gel-0.3 m e-aminocaproic acid system. At pH 3.1, in acrylamide gel electrophoresis, Fraction III plasminogen showed two groups of components, whereas Fraction III2,3 plasminogen showed a single component similar to the minor, faster moving component found in Fraction III plasminogen. Analyses and isolation of the individual isoelectric forms from Fractions III and III2,3 plasminogens were carried out with isoelectric focusing methods. Sixty-six per cent of the plasminogen isolated from Fraction III had isoelectric points between pH 6.2 and 7.2 (six forms). The specific activity of these forms was found to be 20 to 21 casein units per mg of protein. Each of these forms contained a major, and a minor electrophoretic component in slab acrylamide gel electrophoresis in 0.3 m e-aminocaproic acid. Eighty-three per cent of the plasminogen isolated from Fraction III2,3 had isoelectric points between pH 7.2 and 8.1 (four forms). The specific activity of these forms was found to be 22 to 24 casein units per mg of protein. Each of these forms contained two major isoelectric components (doublets). The NH2-terminal amino acid of the isoelectric forms with pI values of 6.2, 6.4, and 6.6 was determined to be glutamic acid. The NH2-terminal amino acid of the isoelectric forms with pI values of 6.7, 7.2, 7.8, and 8.3 isolated from Fraction III2,3, and of the isoelectric forms with pI values of 7.2 and 7.8 isolated from Fraction III was determined to be lysine (and valine). Sedimentation-equilibrium analyses on isolated isoelectric forms showed that the NH2-terminal lysine forms with pI values of 7.2, 7.5, and 7.8, and the NH2-terminal glutamic acid forms with pI values of 6.2 and 6.4 all had the same molecular weights, approximately 80,000. The sedimentation coefficients, s°20,??w, of the Fraction III plasminogen forms were different from the Fraction III2,3 plasminogen forms. Each plasminogen isoelectric form, from pI values 6.2 to 8.5, can be activated with urokinase. The resulting plasmins which were inhibited with l-1-chloro-3-tosylamido-7-amino-2-heptanone, reduced with mercaptoethanol and alkylated with iodoacetic acid, gave Cm-heavy (A) and Cm-light (B) chain derivatives that appeared to be identical with each other when analyzed in acrylamide gel electrophoresis in urea-sodium dodecyl sulfate systems. Cm-heavy (A) and Cm-light (B) chain derivatives isolated from urokinase-activated plasminogen III A appeared to be identical with the same derivatives isolated from urokinase-activated plasminogens 1112,3 A and III2,3 S.

Amelogenins. Purification and partial characterization of proteins from developing bovine dental enamel.

1. Procedures are described for the purification of amelogenin electrophoretic components and their analysis for homogeneity by polyacrylamide-gel electrophoresis at both acidic and alkaline pH values. 2. Most of these components belonged to two main groups, termed the J group and the C group after their major electrophoretic components. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis indicated that, within each group, proteins were of similar size, but the C-group proteins were larger than those of the J group. 3. By sedimentation-equilibrium ultracentrifugation and amino acid analysis, the four J-group components were found to be very small proteins (mol. wt. 5500-3000) and, except for one, similar in amino acid composition. The components of the C group were found to be proteins of moderate size (mol. wt. 16800-16100) with very similar amino acid compositions. A third minor amelogenin group of intermediate size was also found, but not further analysed. Details of the results of the ultracentrifuge studies are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50014 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. 4. Two of the J-group components were similar to amelogenins isolated by other workers. 5. All amelogenins analysed were rich in proline, glutamic acid, histidine and methionine, and contained no half-cystine. Their amino acid compositions, combined with their molecular weights, serve to distinguish the amelogenins from both collagens and keratins.

Naturally occurring crystals in the potato: Isolation and identification as a protein

Cubical crystals occurring naturally in potato tubers were isolated and identified as protein. The amino acid composition was characterized by a relatively large amount of lysine and a near absence of cysteine/cystine. The major electrophoretic component had an approximate molecular weight of 77,000. The preparation exhibited trace proteolytic activity and a weak inhibition of α-chymotrypsin.

Chemical and immunological characterization of the electrophoretic components of the Fc fragment of human immunoglobulin G

The major electrophoretic components of Fc prepared by plasmin digestion of human IgG ( γ 1 subclass) have been isolated and their physical, chemical and biological properties examined. The individual components showed the same amino acid composition, the same amino terminal residue, threonine, and were shown to have the same sedimentation coefficient and molecular weight by ultracentrifugal analysis. Differences in the hexose and sialic acid content of the individual components were shown not to be basis of the heterogeneity. Determination of amide nitrogen by two independent methods revealed differences which could account for the differences in mobility of the Fc components. No differences were found in the ability of the individual components to fix complement or to participate in cutaneous anaphylaxis in guinea pigs.

Genetic control of the major electrophoretic component of rat plasma esterase.

Genetic control of the major electrophoretic component of rat plasma esterase.

Electrophoretic evidence for human prolactin.

ABSTRACT A major electrophoretic component that was active in the pigeon crop sac assay was found in the pituitary gland of a pregnant woman. A similar component was seen in the pituitary of a woman who had been receiving estrogen. The component was present in only small amounts in an ovariectomized woman, in women after menopause, and in males.

Assembly of bacteriophage T4 tail fibers: II. Isolation and characterization of tail fiber precursors

Complete T4 tail fibers and four precursor structures (half fibers) were purified from lysates of mutant-infected Escherichia coli cells, using serum blocking assays for tail fiber antigens to monitor the isolation procedures. Each of the final preparations showed a single major electrophoretic component on polyacrylamide gels. The purified structures, designated whole fibers and A, BC′, BC, and C half fibers on the basis of their antigenic determinants, were further characterized by electron microscopy and assays for ability to produce viable phage in vitro when combined with appropriate extracts of mutant-infected cells. Purified whole fibers are similar in dimensions and appearance to the tail fibers seen on complete phage, and can be attached quantitatively to tail fiberless particles in vitro to produce active virus. The whole fiber is composed of one A and one BC′ half fiber, each about 690 Å in length and 45 Å in width, joined end to end at an angle of about 160 °. The isolated A half fiber carries a knob at one end, which is also visible at one end of the whole fiber. Since the A half is proximal to the tail in the completed phage, the knob presumably represents the point of tail fiber attachment to the baseplate. The A half fiber may not be a normal intermediate in whole fiber assembly, since it is not visible in most crude extracts containing A antigen, and retains little in vitro activity through the purification procedure. The BC′ half fiber, by constrast, appears to be the normal precursor of the distal half of the whole fiber; it is visible in crude extracts and retains its in vitro activity in the purified state. BC, the probable precursor of BC′, is inactive in vitro when purified but indistinguishable from BC′ in dimensions and serological properties. C, the probable precursor of BC, is also inactive in vitro when purified. Besides lacking the B antigen it is shorter than BC, being only 560 Å in length.

Kinetic study of the deamidation of growth hormone and prolactin

1. 1.|Pituitary growth hormore and prolactin are converted to more acidic electrophoretic components when they are allowed to stand in an alkaline medium. A quantitative study of this instability was made by measuring the first-order rate constant for the conversion of the major electrophoretic component to the faster-migrating bands. Changes in concentration of the components were determined by colorimetric assay of the stained disc-electrophoretic bands. 2. 2.|An increase in temperature, pH or ionic strength of the solution, or use of urea, increased the rate of alteration of bovine growth hormone, ovine prolactin and human growth hormone. Urea not only accelerated the conversion, but also increased the number of converted forms. 3. 3.|The rate constants for the conversion were greatest for ovine prolactin under all conditions. Human growth hormone was generally more stable to alteration than ovine prolactin. Bovine growth hormone was the least susceptible to conversion. 4. 4.|The rate of liberation of NH 3 from the hormones at pH 10 correlated with the rate of conversion of the major electrophoretic component. The data were consistent with the assumption that each faster-migrating component was formed by loss of one mole of NH 3. 5. 5.|The multiple disc-electrophoretic forms of ovine prolactin were analyzed by isoelectric focusing in carrier ampholytes. The principal band behaved as a single component, whereas each of the faster-migrating bands was resolved into at least two components. 6. 6.|It was concluded that increased electrophoretic heterogeneity of the hormones, in the absence of proteinases, can be attributed to a nonenzymic deamidation.

Growth hormone activity in man of chymotryptic digests of bovine growth hormone.

SUMMARY Bovine growth hormone (BGH) digested with chymotrypsin showed a significant retention of biological activity after the hydrolysis of six bonds and the formation of 20% non-precipitable nitrogen. Disk electrophoresis of ½ and 3 hr. digests demonstrated complete loss of the major electrophoretic component of BGH with the appearance of one or more components of greater anodal mobility. Administration of the chymotryptic digests of BGH to three hypopituitary patients resulted in an anti-insulin response, aggravation of diabetes mellitus or anabolic effects.

Analysis of histones during organogenesis

1. 1. Histones from adult and embryonic chicken brain, liver, and dorsal skin with feather organ were studied at different developmental stages. Electrophoretic analysis in starch gel revealed seven major electrophoretic components similar to mammalian histones. The electrophoretic patterns were practically identical for all tissue analyzed. 2. 2. The amino acid composition of embryonic histones did not differ from that of adult tissues. In general, the histones from various tissues of chicken contained slightly less lysine and more dicarboxylic amino acids than the unfractionated calf thymus histone. As differentiation progressed an increase was seen in basic amino acids, especially in embryonic dorsal skin histones. 3. 3. The NH 2-terminal amino acids of embryonic and adult chicken histones were alanine (34.3–39.2 per cent) and proline (55.7–60.3 per cent); these two amino acids in similar quantities have been found in most mammalian histones, e.g. calf thymus. 4. 4. It is concluded that histological differentiation and organogenesis in chicken embryonic development is not accompanied by major qualitative changes in histone composition.