Technique Of Starch-gel Electrophoresis Research Articles

Immunologic properties of alkaline phosphatase from human placenta and membranes at delivery

Alkaline phosphatase from human placenta and placental membranes (chorion and amnion) were studied by vertical starch gel electrophoresis and immunologic techniques. By starch gel electrophoresis, it was shown that the mobility of placental AP differed from that of the chorionic and amniotic AP. Similarly, it could be shown by immunoelectrophoresis that chorionic membrane AP migrated more slowly than did placental AP. Using antisera against chorionic AP and placental AP, it was found that placental and chorionic AP are antigenically very similar, if not identical, whereas amniotic membrane AP seems to be antigenically more deficient. All three enzymes were heat stabile.

Studies on biochemical polymorphism in bovines. I. Haemoglobin variations inHariana breed of cattle

The haemoglobin variants in 371Hariana and 39Hariana cross with local breed (Desi) were studied using the starch gel electrophoresis technique. Five different haemoglobin types were observed viz. HbBB, HbBG, HbBA, HbCA and HbAA. Out of these, HbBG has not been reported previously in Indian cattle. The age and sex were found to have no significant effect on the occurrence of haemoglobin types. The population was found to be in Hardy-Weinberg equilibrium.

Metabolic and electrophoretic studies of rat liver sorbitol dehydrogenase.

The effect of various factors on the activity of rat liver sorbitol dehydrogenase was investigated. The activity of the enzyme (expressed per gram of tissue) was similar in the livers of adult male and female rats and was not affected by adrenalectomy, cortisone injection, or hypophysectomy. Different modes of administration of sorbitol also produced little change in activity. The enzyme activity declined significantly during 1 week of starvation. Activity was low in foetal liver and rose gradually during the postnatal period to reach adult levels by approximately 40 days after birth. Sorbitol dehydrogenase activity in the moderately well-differentiated Morris hepatoma 5123 T.C. was studied and found to be approximately 40% of that of normal liver. Using the technique of starch-gel electrophoresis in sodium borate, the rat liver enzyme was found to occur in multiple molecular forms. The electrophoretic pattern of the rat liver enzyme in sodium borate was not affected by a variety of metabolic conditions and was also identical in the hepatoma. From a study of various factors influencing the electrophoretic separation of rat liver sorbitol dehydrogenase, it is concluded that the multiple molecular forms are similar species of the enzyme and that interaction with a starch–borate complex may be important in their resolution.

Comparison of enzymes of twelve species of mosquitoes.

Enzyme comparisons of 12 species of mosquitoes (Diptera: Culicidae) were undertaken using the technique of starch gel electrophoresis. The 4th-stage larvae of 10 species of Aedes, 1 species of Culex, and 1 species of Anopheles were analyzed for acetate and butyrate esterases, alkaline and acid phosphatase, malic and lactic dehydrogenase. Species-specific patterns for all 12 species were obtained with both esterase substrates and for alkaline phosphatase. Aedes aegypti (L.), and A. mascarensis (McGregor) share the same pattern for acid phosphatase and malic dehydrogenase. Both species belong in Group A, subgenus Stegomyia. A. albopictus (Skuse), A. pseudoscutellaris (Theobald), and A. scutellaris (Walker) have identical lactic dehydrogenase patterns; all occur in Group C, Stegomyia. The overall patterns, i.e., obtained with all 6 substrates, were species specific but no clear generic differences were evident. This fact was probably a result of the small sampling of Culex and Anopheles. Inter-specific relationships were examined by means of paired affinity indices and polygonal graphs. These instruments provided the basis for the following taxonomic inferences. A. pseudoscutellaris which is morphologically similar to A. scutellaris and supposedly more closely related to it on a phylogenetic basis, shows a higher degree of biochemical relationship to A. albopictus. On the basis of available data A. triseriatus (Say) seems to be the “biochemical type species” of the subgenus Finlaya. The placement of A. sierrensis (Ludlow) in the subgenus Finlaya on the basis of egg type was reaffirmed on a biochemical basis.

The release of Fc′ and pFc′ fragments from human immunoglobulin G following enzymic digestion: A time study

The techniques of high-resolution gel filtration, immunoelectrophoresis and starch-gel electrophoresis have been used to investigate the enzymic cleavage of human immunoglobulin G (IgG) as a function of time. In particular, the appearance and fate of Fc′ fragment (produced by papain) and pFc′ fragment (produced by pepsin) have been studied. These related fragments, which are located in the C-terminal part of the Fc fragment of the IgG molecule, are relatively resistant to enzymatic degradation whereas the N-terminal portion of the Fc fragment is rapidly degraded. Prolonged exposure to enzyme eventually breaks down both of these fragments but leaves the antibody-binding fragments (Fab or F(ab′) 2 fragments) essentially intact.

Regulating factors in the transfer of iron across the rat placenta.

The effect of maternal blood loss on the iron uptake of the foetectomized placenta has been studied. Normal pregnant rats were used as controls. It is suggested that the rate of maternal blood flow and plasma iron concentration primarily controls placental iron uptake independent of the presence of a foetus. Starch gel electrophoresis and fluorescent antibody techniques have been employed to investigate foetal rat synthesis of transferrin. The onset of synthesis at the commencement of the last third of gestation is probably important in creating a viable system for foetal plasma iron turnover which, under the control of the foetal erythroid marrow, is responsible for the high rate of iron transfer from the placenta to the foetus.

Isozymes of alkaline phosphatase: A report on an autoradiographic method of demonstration

The use of techniques of 45Ca autoradiography and starch-gel electrophoresis presents the investigator with a means of demonstrating the isozymes of alkaline phosphatase. This study indicates that it is a sensitive, workable method which should be applicable to specific physiologic phosphate substrates.

Muscle lactate dehydrogenase isoenzymes in hereditary myopathies

Muscle lactate dehydrogenase (LDH) isoenzymes have been studied in fresh biopsy specimens of superficial limb muscles from patients with various types of muscular dystrophy and the results compared with the findings in normal skeletal muscle. Using the technique of vertical starch-gel electrophoresis a significant reduction in the proportion of LDH-5 was found in 16 out of 23 (70%) of the muscle specimens from patients with muscular dystrophy. The alteration in isoenzyme pattern was not specific for any particular type of muscular dystrophy. When the results from all the patients were combined there was a significant negative correlation between the proportion of LDH-5 and the degree of histological abnormality. In a few cases, however, the proportion of LDH-5 was normal even though the muscle was affected clinically and the histology was abnormal. In the severe Duchenne type of muscular dystrophy LDH-5 was demonstrable when the concentration of the muscle homogenate was adjusted before electrophoresis to contain the same amount of enzyme activity as normal muscle. The reduction in the proportion of M subunits of LDH (referred to as LDH-M) in dystrophic muscle has been confirmed using the technique of urea inhibition. The rate of disappearance of LDH-M in stored homogenates was no greater in dystrophic muscle than in normal muscle, it therefore seems unlikely that there is selective destruction of LDH-M in dystrophic muscle. Interpretation of biochemical findings in the later stages of the disease is difficult because the muscle is invaded by fibroblasts and macrophages which, it has been shown, contain a preponderance of LDH-5.

Isoenzyme polymorphism in flowering plants-VIII. : Isoenzyme variations in tissues of barley seedling

The isoenzyme characteristics of several enzymes in barley ( Hordeum vulgare) var. Blanco Mariout, in relation to tissue differences at an early stage of plant development were studied. Extracts of root, coleoptile, and the first leaf of 8-day-old seedlings were analyzed by the use of starch and polyacrylamide horizontal gel electrophoresis techniques. The isoenzyme patterns of the following enzymes were studied: peroxidases, catalases, esterases, acid and alkaline phosphatases, leucine aminopeptidases, amylases, and glutamic-oxalacetic transaminases. The data regarding the similarities and differences in the isoenzyme composition of the enzymes in the three tissues are presented.

Heat Denaturation of Bovine β-Lactoglobulins and Relevance of Disulfide Aggregation

The primary and secondary stages of the heat denaturation of bovine β-lactoglobulin A have been examined using starch gel electrophoresis and sedimentation velocity techniques. The primary reaction results in the formation of a series of molecular aggregates through the establishment of intermolecular disulfide bonds. A much heavier component is formed during the secondary reaction, but disulfide bonds are not involved in its formation. The results of turbidity experiments indicated that the susceptibility of the genetic variants of β-lactoglobulin to heat denaturation is in the order β—C>β—B>β—A. The relative importance of the formation of disulfide bonds during the aggregation of each variant is discussed. A mechanism describing the behavior during heat treatment is presented.

The α1-acid glycoprotein variants of certain Caucasian patients

α 1-Acid glycoprotein was isolated in homogeneous state from sera of 44 Caucasian patients with various diseases. The resulting glycoprotein preparations were treated with neuraminidase to cleave off their sialyl residues in order to permit starch gel electrophoresis at pH 5 for the identification of the variants. The relative distribution of the three types of α 1-acid glycoprotein variant patterns was found to be very similar to that of normal Caucasian adults. This finding may be considered to be consistent with the concept of the genetically determined transmission of the α 1-acid glycoprotein variants. The α 1-acid glycoprotein preparations were analyzed by starch gel electrophoresis, immunoelectrophoresis and double diffusion techniques. They appeared homogeneous, revealed apparent electrophoretic mobilities identical to that of pooled normal α 1acid glycoprotein and gave precipitin lines which fused completely with each other and with the precipitin line of normal α 1-acid glycoprotein. The sialic acid content of the α 1-acid glycoprotein preparations was essentially identical to that of the acid glycoprotein derived from pooled normal blood. The seromucoid fraction and the α 1-acid glycoprotein levels of these patients' sera, however, showed a wide range and averaged values that were considerably above the normal.

Amino-acid Composition of α s 1 -Casein D

THE genetic polymorphism of αs-casein was first demonstrated by Thompson et al.1 using the technique of starch-gel electrophoresis. Since that time three genetically controlled variants have been found, which have been denominated as -casein A, B and C in order of their decreasing electrophoretic mobility.

Natural Variation in the Blood Proteins of White-Tailed Deer

Starch gel electrophoretic patterns of blood from 200 white-tailed deer (Odocoileus virginianus), mostly from the DeSoto Bend area of the Missouri River on the Io*a-Nebraska border, disclosed two hemoglobin patterns and three presumed transferrin patterns which were compared with each other and with sex and age. This technique disclosed presumptive genetic variation and might be used with blood grouping or alone to characterize different populations of one species or even to estimate mixtures of stocks. To test the feasibility of identifying possible strains or subspecies of deer, the technique of starch gel electrophoresis was applied to 200 blood samples collected for other purposes from wild white-tailed deer in December of 1962 and 1963. The horizontal starch gel electrophoresis was used as described and demonstrated by Mikael Braend to the senior author (1962); it much resembled the method of Smithies (1955) and Poulik (1957). Another detailed description of the technique is given by Manwell (1963). Almost all parts of the serum as well as the hemoglobin (Ingram 1963) can show patterns which vary among individuals within a species, prealbumins (pre), Kristjansson (1963), Shreffler (1964); albumins, Braend (1964), Desborough and Irwin (1964); postalbumins (pa), Gahne (1963); beta globulins or transferrins (Tf), Smithies (1955), Parker and Beam (1962), and Braend and Stormont (1964); and haptoglobins (Hp), Giblett (1961). Genetic studies of these variations wherever made so far have shown simple, or easonably simple, hereditary control (codominance of alleles at one or two loci for each category of variation, Smithies and Hickman (1958), Popp (1963), and previously cited references). One may assume, even in the absence of family data, that similar variation disclosed in related species is likely to have a simple genetic control. Besides disclosing presumptive genetic variation in wild species of animals, such techniques, used with blood grouping (Marr and Sprague 1963) or alone, might enable one to characterize different populations (strains or subspecies) of one species and even to estimate mixtures of stocks. Deer blood from four geographical areas was checked. Most samples, however, came from the DeSoto Bend area of the Missouri River. Deer from this area may be of the subspecies O. v. dacotensis, since deer reintroduced into this area are reported to have been secured from northwestern Nebraska in the 1880's. Samples from northeast Iowa (Lansing) are likely to be of the northern variety (0. v. borealis); the samples from Lucas County in south-central Iowa and 'Journal Paper J-4953 of the Iowa Agricultural and Home Economics Experiment Station, Ames. Projects 1551 and 1395. A joint contribution of the Department of Genetics, Iowa State Unive sity, and Iowa Cooperative Wildlife Research Unit, which is sponsored jointly by Iowa State University, the Iowa State Conservation Commission, the Bureau of Sport Fisheries and Wildlife, and the Wildlife Management Institute. 2 Leader, Iowa Cooperative Wildlife Research Unit, Ames.

Wheat proteins. II. Changes in the protein composition of Triticum vulgare during the life cycle of the plant.

AbstractChanges in the wheat protein system, particularly endosperm proteins, during the life cycle of the plant have been followed using a starch gel electrophoresis technique. Fractions of low electrophoretic mobility were progressively degraded during germination and progressively synthesised towards the end of the ripening period, indicating their probable role as storage proteins of the endosperm. In contrast, fractions of higher mobility were less affected during germination and were utilised at a later stage. Similarly, these components were synthesised first during ripening and remained at a relatively constant level during the build‐up of apparently high molecular weight material. On the information available it was not possible to determine whether the fast‐moving fractions represented the enzymes of the system or were precursors of the larger components.

ABNORMAL HAEMOBLOGIN IN SHEEP.

IN man there exists a large number of abnormal genetically determined haemoglobins. To our knowledge no abnormal haemoglobins are yet reported found in domestic animals. When investigating the haemoglobin phenotypes of experimental sheep with the technique of starch-gel electrophoresis an animal having atypical haemoglobin was found.

Multiple forms of enzymes in a cellular slime mold during morphogenesis

Electrophoretic patterns of acid phosphatase, alkaline phosphatase, and nonspecific esterase were investigated in the cellular slime mold Dictyostelium discoideum by means of starch gel electrophoresis and biochemical techniques. Acid phosphatase was shown to be present in all stages of the life cycle of the slime mold and was separated into five individual bands by the methods of zone electrophoresis employed. Alkaline phosphatase was shown to exist in two forms in this organism, and nonspecific esterase was present in ten distinct forms. The greatest number of enzymatic forms as well as the maximum staining by each group of enzymes was invariably detected during the migrating pseudoplasmodium and culmination stages. In contrast, minimal activity was present in the mature spores. It is suggested that differentiation of this slime mold is accompanied by synthesis of additional enzymatic forms at certain periods during development.

Electrophoresis of serum haptoglobins and erythrocyte catalase using capillary blood

The described technique of thin-layer starch gel electrophoresis allows the direct use of whole blood as electrophoretic substrate for the determination of serum haptoglobin and red blood cell catalase types. Capillary blood absorbed into filter paper strips may be applied to the gel surface freshly, or it may be stored at −20° for later use. An exposure to room temperature for from one to several days of blood dried on filter paper strips does not interfere significantly with subsequent electrophoretic results.

DETECTION OF SMALL, FAST-MOVING HEMOGLOBIN FRACTIONS WITH A MODIFIED STARCH-GEL ELECTROPHORESIS TECHNIC.

IN the course of our investigations on small fast-moving haemoglobin fractions we introduced some modifications in the technique of horizontal starch-gel electrophoresis devised by Smithies1.

GENETIC VARIATION OF PHOSPHATASE IN CATTLE SERUM.

A COMBINATION of starch-gel electrophoresis and histochemical staining technique was used to study a variation in the phosphatase of cattle serum. The starch-gel electrophoresis was performed as described by Gahne1. The starch gel was cut in three slices; the bottom slice was used for protein staining and the middle slice was stained for phosphatase in the following solution: 100 ml. acetate buffer, 0.1 M, pH. 5.0, 100 mg sodium α-naphthyl phosphate. 100 mg o-aminoazotoluene diazonium salt, 10 drops of 10 per cent solution of magnesium chloride, 10 drops of 10 per cent solution of manganese chloride, 0.5 g polyvinylpyrolidone, 2 g sodium chloride. The solution is practically the same as that used by Lawrence et al.2. The gels were incubated 16 h at room temperature. Background staining was removed by washing the gels with methanol/water/acetic acid (50 : 50 : 10). The phosphatase activity was recognized by darkly stained zones.

A new transferrin in New Guinea.

IN 1957 Smithies1, using the technique of starch-gel electrophoresis, described inherited variants of human β-globulins. Smithies and Hiller2 established the identity of these β-globulins with the iron-binding protein, trans-ferrin, and this was afterwards confirmed with iron-59 and autoradiography3. During the past few years widespread sampling of human populations has demonstrated the existence so far of 14 transferrins. In order of decreasing mobility in starch-gel electrophoresis these are4: B0, BO–1 B1, B1–2, B2, B3, C, D0, D4 (D0–1), Dmontreal Dchi, D1, D2, D3. The present communication reports another transferrin of the B series.