Identical Electrophoretic Mobility Research Articles

Electrophoretic identity of biologically active pituitary and serum prolactins in the lactating and perphenazine-treated rat.

ABSTRACT Polyacrylamide gel electrophoresis at neutral pH, 0°C, was used to test the identity of prolactin derived from serum and pituitary extracts of rats. Animals were studied after treatment with perphenazine, a prolactin releasing agent, and during post partum lactation. After gel electrophoresis, the position on the gel of prolactin activity was determined by the pigeon crop-sac bioassay. The biologically active prolactin exhibited identical electrophoretic mobility, whether derived from serum or pituitary, of either normal lactating or perphenazine treated rats.

Immunoglobulin abnormalities in the guillain-barré syndrome

In the Guillain-Barré syndrome (GBS), increased concentrations of the immunoglobulins A and M, and less frequently of immunoglobulin G, expressed as a percentage of the total protein concentration were found in cerebrospinal fluid (CSF). The serum concentrations of these 3 immunoglobulins were normal. One or two discrete bands, probably consisting of oligoclonal immunoglobulins, were demonstrated in the γ region of serum and CSF in 8 of the 12 GBS patients investigated. The discrete serum and CSF γ bands have identical electrophoretic mobilities and are probably transient. High relative concentrations of the 3 immunoglobulins in CSF were especially found in specimens obtained early in the course of GBS; they varied independently of the relative albumin concentrations in CSF. These results indirectly point to a synthesis of the immunoglobulins G, A, and M within the central nervous system in GBS. The demonstration of quantitative immunoglobulin abnormalities and of discrete γ bands on electrophoresis is suggested as having diagnostic implications and also indirectly pointing to the involvement of humoral antibodies in GBS.

The effect of temperature acclimation upon succinic dehydrogenase activity from the epaxial muscle of the common goldfish ( Carassius auratus L)—I. Properties of the enzyme and the effect of lipid extraction

1. 1. Succinic dehydrogenase actuvity from the epaxial muscle of 5°C-acclimated goldfish was 1·3–2·2 times (depending upon season) that of 25°C-acclimated goldfish when measured at the same temperature in both crude homogenates and isolated mitochondrial fractions. 2. 2. Lipid extraction (formation of an acetone powder)_ of the particulate enzyme resulted in a three- to fourfold drop in specific activity, a reduction in the E a and a reduction in the absolute K m values for succinate. 3. 3. Electrophoresis of soluble preparations from both 5- and 25°C-acclimated goldfish yielded a single SDH band of identical electrophoretic mobility from each acclimation group. 4. 4. Kinetics studies revealed that the K m for PMS was temperature independent and did not vary with acclimation temperature; particulate and soluble preparations were characterized by identical K m values for PMS. 5. 5. There was a direct reltionship between the K m for succinate and the assay temperature in both soluble and particulate preparations which was identical for both acclimation groups; the magnitude of the K m temparature response was reduced by lipid extraction. 6. 6. Inhibition of the soluble enzyme by oxaloacetic acid was extremely temperature dependent as evidenced by a twelve- to fifteenfold increase in i upon a drop in assay temperature from 25 to 5°C. 7. 7. Goldfish SDH† was characterized by a very steep pH-activity profile in the physiological range. 8. 8. E a measurements suggested a positive correlation between habitat temperature and activation energy. 9. 9. These results were interpreted as indicating that SDH normally functions as a lipoprotein complex within the inner mitochondrial membrane and exhibits properties which maintain temperature-independent catalytic function in response to short-term and prolonged temperature fluctuations.

Comparison of mammalian mitochondrial ribosomal ribonucleic acid from different species.

Mitochondrial ribosomal RNA species from mouse L cells, rat liver, rat hepatoma, hamster BHK-21 cells and human KB cells were examined by electrophoresis on polyacrylamide-agarose gels and sedimentation in sucrose density gradients. The S(E) (electrophoretic mobility) and S values of mitochondrial rRNA of all species were highly dependent on temperature and ionic strength of the medium; the S(E) values increased and the S values decreased with an increase in temperature at a low ionic strength. At an ionic strength of 0.3 at 23-25 degrees C or an ionic strength of 0.01 at 3-4 degrees C the S and S(E) values were almost the same being about 16.2-18.0 and 12.3-13.6 for human and mouse mitochondrial rRNA. The molecular weights under these conditions were calculated to be 3.8x10(5)-4.3x10(5) and 5.9x10(5)-6.8x10(5), depending on the technique used. At 25 degrees C in buffers of low ionic strength mouse mitochondrial rRNA species had a lower electrophoretic mobility than those of human and hamster. Under these conditions the smaller mitochondrial rRNA species of hamster had a lower electrophoretic mobility than that of human but the larger component had an identical mobility. Mouse and rat mitochondrial rRNA species had identical electrophoretic mobilities. Complex differences between human and mouse mitochondrial rRNA species were observed on sedimentation in sucrose density gradients under various conditions of temperature and ionic strength. Mouse L-cell mitochondrial rRNA was eluted after cytoplasmic rRNA on a column of methylated albumin-kieselguhr.

A Lactate Dehydrogenase Specific to the Liver of Gadoid Fish

Comparative studies on tissue extracts of haddock, cod, and related species in the suborder Gadoidei indicated the presence of a lactate dehydrogenase specific to liver tissue. Genetic and evolutionary variation of the lactate dehydrogenase isozymes in these fishes indicate that the liver isozyme is encoded at a structural gene locus genetically distinct from the loci encoding the heart and muscle isozymes. The properties of purified liver, heart, and muscle lactate dehydrogenases from haddock were compared to investigate their relationship. The haddock liver enzyme is like other lactate dehydrogenases in having a molecular weight of 140,000 with a subunit size of 35,000. The liver enzyme is distinguished from the heart and muscle isozymes by its greater resistance to heat inactivation. The gadoid heart and muscle isozymes do not hybridize with each other in vivo or in vitro, but the liver enzyme can hybridize with both in vitro, although not in a random fashion. The catalytic properties of the haddock liver and heart isozymes are very similar; they are distinct from the muscle isozyme in having a greater affinity for both lactate and pyruvate and in being inhibited at high pyruvate concentrations. The liver enzyme showed no unusual substrate specificities. Ironically, the liver enzyme can be mistaken for the muscle enzyme since both have nearly identical electrophoretic mobilities. The catalytic properties of the haddock heart and liver enzymes suggest that these two may be homologous and that both might be homologous to the heart-type lactate dehydrogenase of the higher vertebrates. The immunological crossreactivity of these two with each other and with chicken heart lactate dehydrogenase provides confirmation for this hypothesis. The immunological data suggest a considerable degree of sequence homology between the heart and liver lactate dehydrogenases of gadoid fishes. From evolutionary considerations, it appears that the divergence of the heart and liver loci from an ancestor heart-type locus is a relatively recent event, perhaps arising concurrently with the evolution of the gadoid fish into a distinct lineage.

Human Phosphoglycerate Kinase: II. STRUCTURE OF A VARIANT ENZYME

Abstract A human phosphoglycerate kinase variant, associated with faster anodal electrophoretic mobility in starch gel, was purified and crystallized from red blood cells of subjects in New Guinea. An apparently identical variant enzyme was also purified from a male in the Samoa Islands. The variant enzyme had the same molecular weight, 50,000, and the same specific enzyme activity, 700 units per mg, as the wild type enzyme. The isoelectric point of the variant enzyme, determined by the isoelectric focusing method, was pH 4.7 as compared with pH 7.9 for the normal enzyme. Although active variant enzyme moved toward the anode much faster than the normal enzyme in starch gel electrophoresis using a Tris-citrate buffer system at pH 7.5, no significant difference was found in electrophoretic mobility of the enzymes in other buffer systems without citrate. Lyophilized preparations of the normal and variant enzyme were enzymatically inactive, showed identical electrophoretic mobility in polyacrylamide gel electrophoresis using Tris-citrate buffer at pH 7.5, and had identical isoelectric points. These findings suggest that the fast electrophoretic mobility and the acidic isoelectric point of the variant enzyme are not due to intrinsic charge differences caused by the amino acid substitution, but due to a secondary alteration of the protein charge presumably induced by association with citrate. In fact, an electrically neutral amino acid substitution, from threonine in the normal enzyme to asparagine in the variant enzyme, was suggested by peptide mapping of their tryptic digests. The phosphoglycerate kinase variant found in a male from the Samoa Islands was identical with the variant enzyme found in New Guinea.

Synthesis and secretion of gammaglobulin by lymph node cells: the acquisition of carbohydrate residues of immunoglobulin in relation to interchain disulfide bond formation (heavy and light chains-murine myeloma-mannose-glucosamine-galactose).

Acquisition of carbohydrates in the disulfide-linked heavy (H) and light (L) chain molecules of murine myeloma (ADJPC5), i.e., HH, HHL, and LHHL, was investigated. That some mannose and glucosamine residues are acquired by immunoglobulin precursor molecules was demonstrated by the detection of glucosamine and mannose in HH, HHL, and LHHL. In contrast, galactose was observed solely in LHHL molecules, which have an identical electrophoretic mobility to the secreted product. Furthermore, as judged from cells incubated with [(3)H]leucine, the more juvenile molecules HH and HHL were predominant in the rough microsome fraction, whereas LHHL was the principal molecular species in the smooth microsome fraction. Findings of this type were not observed in rabbit lymph node cells. Thus, galactose, as well as mannose and glucosamine, were found in the more juvenile molecule known for this species (HL). Moreover, the ratio of HL:LHHL, as judged from cells incubated with [(3)H]leucine, was about the same in rough and smooth microsomes.

Studies on proteins of animal ribosomes VIII. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins of rat liver

Electrophoresis was carried out in the first dimension in 5% gels at pH values of 8.9 in the gel and 8.3 in the buffer, and in the second dimension in 15% gels at pH values of 4.5 in the gel and 2.0 in the buffer. Total ribosomal protein was separated into more than 60 spots. Increased resolution efficiency was achieved by electrophoresis of subunit proteins. 34 protein spots specific for the large subunit and 27 spots specific for the small subunit were recognized. In addition there are 15 spots with identical electrophoretic mobility in either protein mixture. Altogether, 76 cationically moving proteins were demonstrated.

The isolation and characterization of proteins from the left colleterial gland of the cockroach, Periplaneta americana (L.)

Previous work has established that the cockroach egg-case is constructed of proteins which are cross-linked by reaction with the oxidation products of ortho-diphenols. In the present study the predominant proteins of the egg-case were isolated, before they had been crosslinked, from the secretion of the left colleterial gland. They can be separated into waterinsoluble proteins in the form of globules which can be seen in the electron microscope, and water-soluble proteins. When these two fractions were separated from each other by sucrose density gradient centrifugation, phenol oxidase activity was found to be associated with the water-soluble proteins. Electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulphate shows that the water-insoluble protein fraction contains two major and one minor proteins; the water-soluble protein fraction contains three major proteins. The molecular weights of these proteins were determined from their mobilities on electrophoresis by this method. The proteins were separated by gel filtration chromatography and the homogeneity of the individual fractions was tested by electrophoresis and iso-electric focusing on polyacrylamide gels. The amino acid composition of each protein fraction was obtained. The two major water-insoluble proteins have high contents of glycine, tyrosine and leucine, and two of the water-soluble proteins have high contents of glycine, tyrosine and valine. The minor protein of the water-msoluble fraction and the third wrater-soluble protein have identical electrophoretic mobilities and relatively high contents of Lysine, proline and valine. The compositions of the four proteins with high contents of glycine show some resemblances to those of silks but are significantly different from anything previously described. The high tyrosine contents of these four proteins suggest that tyrosyl residues may be involved in the formation of the cross-links.

Tissue haemoglobins from the gastropod molluscs lunatia heros say and busycon canaliculatum L.

1. 1. Busycon canaliculatum nerve haemoglobin and heart and radular muscle myoglobins have the same or very similar molecular weights (about 32,000) and absorption spectra; the corresponding globins have identical electrophoretic mobilities in 8 M urea. 2. 2. The native Busycon canaliculatum haemoglobins are dimeric and are cleaved to monomeric subunits by reaction p-hydroxymercuribenzoate. 3. 3. Lunatia heros nerve haemoglobin and radular muscle myoglobin have the same or very similar molecular weights (about 15,000) and absorption spectra, and identical electrophoretic mobilities. 4. 4. It is probable that Busycon canaliculatum and Lunatia heros possess only a single speciesspecific type of haemoglobin in their tissues.

The low molecular weight RNAs of Rous sarcoma virus: I. The 4 S RNA

Chromatography of Rous sarcoma virus RNA on methylated albumin-kieselguhr has allowed the identification of four classes of intravirion RNA: a substantial amount of 4 S RNA, a discrete 7 S component, small quantities of 18 and 28 S RNA, and the 70 S RNA of the viral genome. The 4 S RNA's of the virus and its host cell have identical electrophoretic mobilities in 10% polyacrylamide gels and are methylated to the same extent. However, significant differences in nucleotide compositions are detectable. The virus apparently does not contain RNA corresponding to any of the other low molecular weight species of cellular RNA. The data are in accord with previous suggestions that RNA tumor viruses contain tRNA acquired from the host cell during assembly, but also indicate at least minor differences in composition between the cellular and viral 4 S RNA populations. The results of reconstruction experiments suggest that the viral 4 S RNA is not simply a contaminant derived from cellular debris.

Comparison of In Vitro and In Vivo Labeling of Virus-Induced L-Cell Interferon

Preparations of NDV(uv)-induced L-cell interferon were labeled in vitro with (125)I and (3)H gas, or in vivo through incorporation of amino acids-(3)H during synthesis. Prior to purification, more than 90% of the interferon titer was lost during in vitro labeling by either procedure, whereas 34% of the initial activity of in vivo-labeled material was preserved during preparatory handling. Purification by carboxymethyl-Sephadex chromatography and electrophoresis in polyacrylamide gels was about 100-fold, and electrophoretic profiles revealed close concordance between isotopes and interferon titers in all instances. Noninterferon proteins from control cells, although less extensively labeled with tritium during synthesis than proteins from interferon-producing cells and released in lesser amounts, also contained components of identical electrophoretic mobility and distribution in acrylamide gels as interferon. The highest specific activity (6 x 10(6) U/mg protein) but lowest cpm per interferon unit ratio (0.3) were exhibited by in vivo-labeled interferon. The advantage of better isotope incorporation through in vitro labeling techniques was largely offset by extensive losses in interferon activity.

Immunocemical characterization of submicrosomal rat liver esterases

Antigens from 3 submicrosomal rat liver fractions (R, Sa and Sb) were investigated for esterase activity in immunoelectrophoretic experiments with rabbit antisera against each of the subfractions. The esterases were further classified as carboxyl-, aryl-, acetyl- or choline esterases by their sensitivity to inhibition by 10 −3–10 −4 M DFP, 10 −4 M eserine, 10 −2–10 −4 M PCMB, heat and 10 M urea. The fractions R and Sa contained at least 3 acetyl esterases and 5 carboxyl esterases, while only 1 acetyl esterase and 2 carboxyl esterases were detected in the Sb-fraction. No aryl- or choline esterases were found in any of the fractions. Liver microsomes were shown to have one carboxyl esterase in common with kidney microsomes and one acetyl esterase in common with testes microsomes. By the use of different substrates, it was shown that short chain fatty acids were more easily attacked by the esterases of liver microsomes than long chain fatty acids. The activity of one esterase active antigen classified as carboxyl esterase and occurring in three variants of identical electrophoretic mobility, was strongly incresed in the R- and Sa-membranes by phenobarbital treatmet. In the Sb-membranes, this antigen is entirely missing. Two of the variants were also shown to be present in kidney microsomes where phenobarbital did not provoke any enhancement.

The emergence of antibodies with either identical or unrelated individual antigenic specificity during repeated immunizations with streptococcal vaccines.

Electrophoretically monodisperse antibody components in rabbit antisera to the carbohydrates of the Groups A and C streptococci have been examined for their individual antigenic specificity. In these antibody components which were isolated by preparative electrophoresis, individual antigenic specificity was confined to the specific antibody and was absent in the nonantibody gamma-globulin. Radioprecipitation experiments and the use of immune absorbent columns constructed from goat anti-antisera, which had been absorbed with fraction II, revealed that all the specific antibody in an electrophoretically monodisperse component was reactive with the homologous anti-antibody. Antibodies with either identical or distinct individual antigenic specificities may occur in the same rabbit with repeated immunizations. Antibodies with identical antigenic specificity had identical electrophoretic mobility, whereas antibodies with unrelated antigenic specificities had distinct electrophoretic mobilities. In the interval between immunizations, if antibody to the carbohydrate antigen was absent, there was no detectable antibody with individual antigenic specificity.

Electrophoretic, solubility and thermostability differences in proteins of eye lens nuclei from two closely related fish species, the yellowfin tuna and the bigeye tuna

1. 1. Electrophoretic separation of protein from lens nuclei of yellowfin and bigeye tunas produced species-specific patterns. 2. 2. Certain of these proteins from the yellowfin and bigeye had identical electrophoretic mobilities but differed in solubility. 3. 3. In both tuna species, proteins from heated extracts revealed an increasing thermostability with increasing electrophoretic mobility and molecular weight. Proteins of bigeye, however, were more heat-stable than those of yellowfin. 4. 4. The electrophoretic, solubility and thermostability data indicate that the low molecular weight nuclear lens proteins are less alike than those of high molecular weight in related species.

Sex difference in alcohol metabolism; androgenic steroid as an inducer of kidney alcohol dehydrogenase

Available genetic evidence on a number of other species shows that alcohol dehydrogenase (ADH: 1.1.1.1) seen in liver and kidney of vertebrates is specified by the same gene locus. Also in the case of man and the mouse, ADH from liver and kidney show identical electrophoretic mobilities. Yet, in development, ADH appears much earlier in liver than in kidney, and a twofold sex difference is seen in the amount of ADH contained in adult kidney of mice and man: Kidney ADH activity of adult females is about one‐fourth and that of adult males is about one‐half of the liver ADH activity.When castrated adult male and female mice are given 10 mg testosterone, kidney ADH activity rises to about the same level as liver ADH.On the basis of the above, we postulate that the ADH locus of kidney but not of liver is under the control of a secondary repressive genetic regulatory mechanism. This genetic block on transcriptional and translational activity of the ADH locus is removed by androgenic steroid which serves as an inducer. As long as this block is completely removed, the ADH locus of kidney functions at about the same innate rate as the ADH locus of liver.

The two-dimensional electrochromatography of uroproteins

50 samples of urine of patients who excreted uroproteins in quantities of 0.10–0.45 gm% and 1 urine sample with paraproteins as well as their corresponding sera were analyzed with the method of two-dimensional electrochromatography on filter paper and by one-dimensional paper electrophoresis. The results of the two-dimensional electrochromatography point to the fact that the chromatographic and the electrophoretic effect of uroproteins is identical with the electrochromatographic effect of seroproteins. Consequently the seroproteins do not undergo any change concerning their electrochromatographic effect during the filtration process through the kidney into the urine. This fact concerning the electrophoretic effect was also simultaneously confirmed with one-dimensional paper electrophoresis. The two-dimensional electrochromatographic separation of uroparaproteins and sero-paraproteins demonstrated the same chromatographic effect but different electrophoretic effects at variance with the one-dimensional electrophoresis, where identical electrophoretic mobilities were found in uro- and in sero-paraproteins.

Phenotypic variations of haptoglobin Johnson types.

Sera from four individuals with haptoglobin Johnson phenotypes were examined. Polyacrylamide gel electrophoresis revealed minor phenotypic variations between the Johnson types of two members from a Czechoslovakian family and of two unrelated Caucasians from the USA. The subtyping procedure after haptoglobin isolation and reductive cleavage showed Hp α J-polypeptide chains with identical electrophoretic mobilities in the four individuals analyzed. In addition to the Hp α J chain two persons had a Hp α 1 S chain and two persons had a Hp α 1 F chain. The two members of the Czechoslovakian family had, furthermore, a third component with an electrophoretic migration rate intermediate between a common Hp α 2 and a common Hp α 1 S chain.

Qualitative studies of the protein in fetal lamb lung fluid.

SummaryThe protein fractions present in fetal lung fluid were determined by means of cellulose acetate and immunoelectrophoresis. Proteins with identical electrophoretic mobility to that of sheep albumin and α globulins were detected in all; prealbumin, beta and gamma globulins were observed in some. An unidentified γ globulin other than γ G-globulin was shown to be present in fetal lung fluid and absent in fetal serum. The α2 globulins in fetal lung fluid were found in a concentration which exceeds that of the serum. The local synthesis of both proteins in the lung is suggested.

Hematology of the ursidae

1. 1. A total of fifty-nine blood samples from fifty-three bears, representing the genera Ursus, Tremarctos, Thalarctos, Selenarctos, Melursus and Helarctos have been analyzed. Measurements made included hemoglobins, hematocrits, red blood cell counts, and total white blood cell counts. 2. 2. The erythrocyte indices, including mean cell volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated. Statistically significant differences between species, ages, and seasons were found in the measurements, particularly as reflected in the mean cell volume. 3. 3. Three pregnant females exhibited a 1–3 g per cent decrease in hemoglobin concentration during the last month of pregnancy. The mean red cell volume of newborn cubs of Selenarctos thibetanus and Ursus arctos horribilis was 50 per cent greater than that of the adults. 4. 4. Hemoglobin samples from sixty-six bears were examined by electrophoresis in polyacrylamide gel. A single major hemoglobin, of identical electrophoretic mobility was found in all species and all specimens, including young and newborn cubs.