Gel Electrophoretic Studies Research Articles

Structural and regulatory properties of pyruvate kinase from Pseudomonas citronellolis.

Pseudomonas citronellolis contains a single variety of pyruvate kinase regardless of the substrate on which the organism is grown. The enzyme has been purified to homogeneity and shown by cross-linking and gel electrophoretic studies to be a tetramer of M/sub r/ = 225,000 to 240,000. Unlike pyruvate kinases from yeast, liver, kidney, and red cells, the Pseudomonas enzyme is not affected by fructose 1,6-bisphosphate. However, it is activated by 2-keto-3-deoxy-6-phosphogluconate (KDPG), an intermediate of the Entner-Doudoroff pathway which is utilized for glucose catabolism by this organism. The Pseudomonas enzyme is also activated by ribose-5-P,5'-AMP, and fructose-6-P. Kinetic studies suggest that this group of metabolites may act at a single site with KDPG activation occurring at a separate site. All of the activators function by increasing the affinity of the enzyme for P-enolpyruvate. The latter substrate shows a strong positive cooperativity with the enzyme which is lowered or abolished by the presence of the activators. A strong synergistic effect exists between KDPG and ribose-5-P as exhibited by a mutual lowering of the concentrations required for activation as well as by a more than additive effect on the enzyme's affinity for P-enolpyruvate. Pyruvate kinase from P. citronellolis also differs from most varietiesmore » of the enzyme in that it is not activated by K/sup +/ orNH/sub 4//sup +/, requires high concentrations of Mg/sup 2 +/ and is relatively nonspecific with respect to its nucleoside diphosphate substrate.« less

Genetics of the mammalian phenylalanine hydroxylase system. II. Immunological and two-dimensional gel electrophoretic studies of phenylalanine hydroxylase in cultured normal and mutant rat hepatoma cells.

We have examined 11 previously described cultured rat hepatoma mutants with absent or reduced phenylalanine hydroxylase activity (Choo and Cotton, 1977). Immunological and electrophoretic methods failed to detect any structurally altered protein in these mutants. In nine independently isolated revertants from four different mutants, wild-type protein was regained (or accentuated). This evidence suggests that the mutation involved in these mutants is most likely to be regulatory in nature. These studies have provided three reasons for believing that in cultured rat hepatoma cells one gene codes for a single polypeptide chain, a number of which combine to form the active phenylalanine hydroxylase multimer: (1) Analysis of the purified protein by two-dimensional electrophoresis revealed only a single polypeptide chain. (2) This polypeptide was diminished or undetectable in crude extracts of 11 independently isolated mutants with absent of reduced activity. (3) In none of these 11 mutants was the polypeptide we have designated to be phenylalanine hydroxylase present at normal levels, as would be expected if the mutation were at another locus responsible for a possible second subunit.

ELECTROPHORETIC PATTERNS OF OAT PROLAMINES AND SPECIES RELATIONSHIPS IN AVENA

Starch gel electrophoretic studies on seed prolamines of 17 different di-, tetra-, and hexaploid Avena species were undertaken to determine species relationships and the evolutionary process of the polyploids. Significant intraspecific variation was observed. Electrophoregrams of each species were obtained by superposing all the bands found in the same species and in total, 17 bands were observed. Clear differences were found between species in their patterns, which allowed determination of the bands specific to three genomes (A, C and D). Like the diploids, the tetraploids can be divided into two groups on the basis of electrophoretic patterns: A. barbata and A. magna-A. murphyi. It is evident that A. magna-murphyi group is derived from both the A and C genome species of diploids while A. barbata is derived from the A genome species only. The similarity between A. magna and A. murphyi was also confirmed. The hexaploids were derived from A. magna-murphyi group and a D genome ancestor although the last one is not discovered yet.

Evidence for an Altered Physical State of Membrane Proteins in Erythrocytes in Friedreich's Ataxia

Electron spin resonance, scanning electron microscopic, and SDS-polyacrylamide gel electrophoretic studies of erythrocytes in Friedreich's ataxia have been performed. No alteration in the physical state of membrane lipids, in morphology, or in the staining profile of erythrocytes in Friedreich's ataxia could be demonstrated. An altered conformation and/or organization of proteins in erythrocyte membranes in this disorder was suggested by spin labeling studies (P less than 0.025), favoring the possibility of a generalized membrane abnormality in Friedreich's ataxia. These findings are discussed in relation to other inherited neurological diseases where similar studies have been performed.

Transduction of penicillinase production in Staphylococcus epidermidis and nature of the genetic determinant

Four strains of Staphylococcus epidermidis from clinical sources were capable of serving as donors for the transduction of either penicillinase production, ethidium bromide resistance, or tetracycline resistance. Three typing phages served as transducing phages and, depending upon the combination of transducing phage, donor strain, and recipient strain, the rates of transduction ranged between 10(-5) and 10(-9). In one strain, cotransduction of penicillinase production and ethidium bromide resistance was observed. Although ultraviolet irradiation kinetics indicated that both the tetracycline resistance and the penicillin resistance determinants were located on plasmids, only resistance to tetracycline could be eliminated by growth in the presence of curing agents or at elevated temperature. However, evidence was obtained by agarose gel electrophoretic studies that both the tetracycline resistance and the penicillin resistance determinants are located on separate plasmids in this organism.

Distribution and Characteristics of Aminoacyl β-Naphthylamidase Activities in Wheat Seedlings

Gel electrophoretic studies have revealed that crude extracts from various tissues of wheat seedlings contain two major enzymes capable of hydrolysing aminoacyl �-naphthylamides. A third enzyme which exclusively hydrolyses the �-naphthylamides of the imino acids proline and hydroxyproline has also been demonstrated in wheat leaves. The pH optimum for �-naphthylamidase activity against phenylalanine �-naphthylamide in crude extracts was 7.4. The two major enzymes differ with respect to their substrate specificities; the more anionic enzyme, APl, hydrolyses a relatively narrow range of hydrophobic aminoacyl substrates including the �-naphthyIamides of leucine, phenylalanine, methionine, tyrosine and tryptophan, while the enzyme of lower electrophoretic mobility, AP2, hydrolyses a broad range of substrates. The two enzymes also differ in their sensitivity to the metal chelator 1,10-phenanthroline. The AP2 enzyme from wheat, like that from pea, appears to be a metallo-enzyme, but APl does not show any sensitivity to phenanthroline. The results of developmental studies performed with wheat seedling tissues are consistent with the view that the naphthylamidase enzymes function as aminopeptidases in vivo. A close association was observed between total enzyme activity and soluble protein content, with the highest naphthylamidase activities being found in actively growing tissues.

Photo- and GA3-induced Changes in RNAs in Impatiens balsamina L.

Summary Polyacrylamide gel electrophoretic studies show that new RNA species develop within one day both in the stem and the leaves of Impatiens balsamina L. plants exposed to inductive treatments regardless of whether the induction of floral buds was caused by 8 hr photoperiods (inductive) or by GA 3 applications under 24 hr photoperiods (non-inductive). A band with high electrophoretic mobility also develops but only in the leaves of GA 3 -treated plants under 8 as well as 24 hr photoperiods and may be considered to be associated with extension growth.

Myelin basic protein microheterogeneity in subfractions of rat brain myelin.

Abstract— Myelin subfractions were prepared from adult rat brain by discontinuous sucrose gradient ultracentrifugation. Gel electrophoretic studies at pH 10.6 in the presence of urea revealed differences in basic protein microheterogeneity among subfractions. With increasing myelin density there was a decrease in the most positively charged components of both large BP and small BP. Since these components are the least modified by deamidation and phosphorylation, it seems likely that the heavier myelin subfractions are enriched in the more modified components of the microheterogeneous population of BP. These observed differences may be related to the regulatory processes controlling biosynthesis, organization, and catabolism of BP in CNS myelin.

EMULSIFICATION, FOAMING AND PROTEIN SOLUBILITY PROPERTIES OF DEFATTED SOYBEAN, PEANUT, FIELD PEA AND PECAN FLOURS

ABSTRACTDefatted soybean, peanut, field pea and pecan flours were blended as 8% suspensions (w/v) with distilled water and characterized for emulsion and foam capacity, emulsion viscosity, protein solubility, and gel electrophoretic properties. Flour suspensions were evaluated for these properties at their natural pH, at pH 4.0, 8.2 and after a two‐step sequential adjustment from the natural pH to 4.0 to 8.2. Maximum functionality of suspensions was noted as follows: (a) soybean flour, very thick mayonnaise‐like emulsions (> 160,000 cps) and egg white‐type foams at natural pH; (b) peanut flour, semi‐thick mayonnaise‐like emulsions (70,880 cps) and thick egg white‐type foams after two‐step adjustment to pH 8.2; (c) field pea flour, semi‐thick mayonnaise‐like emulsions (66,240 cps) and medium‐thick foams after adjusting the pH directly to 8.2; and (d) pecan flour, very thick mayonnaise‐like emulsions (> 160,000 cps) after two‐step adjustment to pH 8.2. Protein solubility seems to be more closely associated with improved quality of emulsions and foams formed than with increased quantity of oil or air that could be bound by flour suspensions. Data from gel electrophoretic studies suggested that the major seed storage proteins were important in functionality tests, although other seed constituents, such as carbohydrates, may be equally involved.

Glycopeptide fractions prepared from purified central and peripheral rat myelin

Myelin was purified from rat brain and sciatic nerve after invivo labeling with [ 3H]fucose and [ 14C]glucosamine to provide a radioactive marker for glycoproteins. The glycoproteins in the isolated myelin were digested exhaustively with pronase, and glycopeptides were isolated from the digest by gel filtration on Bio-Gel P-10. The glycopeptides from brain myelin separated into large and small molecular weight fractions, whereas the glycopeptides of sciatic nerve myelin eluted as a single symmetrical peak. The large and small glycopeptide fractions from central myelin and the single glycopeptide fraction from peripheral myelin were analyzed for carbohydrate by colorimetric and gas liquid chromatographic techniques. The glycopeptides from brain myelin contained 2.4 μg of neutral sugar and 0.59 μg of sialic acid per mg total myelin protein, whereas sciatic nerve myelin glycopeptides contained 10 μg of neutral sugar and 3.8 μg of sialic acid per mg total protein. Similarly, the gas-liquid chromatographic analyses showed that the glycopeptides from peripheral myelin contained 4- to 7-fold more of each individual per mg total myelin protein than those from central myelin. Most of the sialic acid and galactose in the glycopeptides from central myelin were in the large molecular weight fraction, and the small molecular weight glycopeptides contained primarily mannose and N- acetylglucosamine . The considerably higher content of glycoprotein-carbohydrate in peripheral myelin supports the results of gel electrophoretic studies, which indicate that the major protein in peripheral myelin in glycosylated while the glycoproteins in purified central myelin are quantitatevely minor components.

Effect of irradiation on electrophoretic properties of enzymes in haemopoietic cells of opossum.

5-month-old male opossums were exposed to 5000 rads whole body 60Co radiation and sacrificed at 16, 40 and 90 h following irradiation. Stract gel electrophoretic studies of dehydrogenases of lactate, malate, 6-phospho-gluconate and glucose-6-phosphate and alpha-esterases and acid phosphatase were conducted on the homogenates of spleen and bone marrow cells. Expressions of LDH and G6PD were affected by irradiation in bone marrow and spleen cells.

Gel electrophoretic studies of proteins in photo-induced and vegetative plants of Impatiens balsamina

In Impatiens balsamina L. var. Rose, a qualitative SD plant, the protein content of the leaves shows an upsurge at the time of physiological induction. The electrophoretic pattern of water soluble proteins, however, does not change except for a new protein that appears in the stem after the plants have received 1 SD cycle.

Regulation of adenylate cyclase from glial tumor cells by calcium and a calcium-binding protein.

A biphasic response to changes in Ca2+ concentration was observed for basal and norepinephrine-stimulated adenylate cyclase activity in homogenates of C-6 glioma cells. The enzyme was stimulated approximately 40% by low concentrations of free Ca2+ (less than or equal to 1 muM) and inhibited to successively greater extents as free Ca2+ concentrations were increased to approximately 100 muM. Ca2+ did not alter the concentration of norepinephrine required for enzyme activation. Homogenates of C-6 cells were separated into particulate and supernatant fractions by centrifugation at 27,000 X g for 20 min. The particulate fraction contained nearly all of the adenylate cyclase activity. This activity was stimulated approximately 40% by the addition of untreated supernatant fraction, by boiled or dialyzed supernatant fraction, and by a homogenous Ca2+-binding protein (calcium-dependent regulator (CDR) prepared from brain. Addition of either the supernatant fraction or CDR lowered the Ca2+ concentration required for maximal stimulation of the adenylate cyclase. The factor in the supernatant fraction which activated the particulate enzyme was subsequently identified in acrylamide gel electrophoretic studies to be CDR. The amount of CDR required for maximal activation of the enzyme was found to be lowered as the Ca2+ concentration in the assay was increased. High amounts of added CDR (100 to 1000 ng) were inhibitory. Use of the monionic detergent, Lubrol PX, to prepare dispersed adenylate cyclase from the particulate fraction resulted in large losses of activity. The resultant preparation of enzyme contained some CDR which could not be removed by chromatography of the preparation on anion exchange columns. Addition of homogeneous CDR to the assay activated the enzyme several-fold at low Ca2+ concentrations. At higher Ca2+ concentrations the enzyme was activated fully by the CDR endogenous to the preparation and added Ca2+. CDR was inhibitory.

Immunological and immunochemical properties of the giant African snail ( Acathina Fulica) hemocyanin

The immunological and immunochemical properties of the giant African snail ( Acathina fulica) hemocyanin were studied and compared with those of the keyhole limpet ( Megathura crenulata) hemocyanin. No 7S antibody could be detected 5 or 10 days after injection of crude Acathina fulica hemocyanin, whilst most of the antibody obtained in sera at 25 days belonged to 7S globulin type. Primary, secondary and hyperimmune antisera to keyhole limpet hemocyanin cross-reacted with the African snail hemocyanin. However, primary, secondary and hyperimmune antisera to the African snail hemocyanin did not cross-react with keyhole limpet hemocyanin. Polyacrylamide disc gel electrophoretic studies revealed that keyhole limpet hemocyanin contained at least 10 different moieties, all of which were antigenic, while the African snail hemocyanin contained about 4 moieties, two of which were strongly antigenic. Examination of the immunochemical behaviour of the African snail hemocyanin under various solvent conditions, showed that it existed in polymeric form in its native state and at pH 7.5, while at pH 8.5 it existed in monomeric form.

Electron microscopic, histochemical and disc gel electrophoretic studies on the deoxycholate soluble proteins from human plantar horny layers.

The matrix proteins of human plantar horny layers were extracted with deoxycholate, purified and examined with the light and electron microscopes, and their histochemical characteristics were investigated. About 44.3% of the total proteins was released into the 270,000 X g supernatant fraction, following incubation with deoxycholate, and the precipitate obtained by dialysing this supernatant fraction consisted of fine granular materials which were found to be electron dense, round material under the electronmicroscopy. Small electron dense dots were seen on the surface of this material. This pellet was positive with Pauly's reagent, toluidine blue and Harris hematoxylin-eosin. However, the material further purified by the molecular sieve chromatography was negative with Pauly's reagent. And it had been proved to be of keratohyalin granules origin by the indirect immunofluorescent study. Another protein fraction which was obtained by the molecular sieve chromatography was positive with diazotized sulfanilic acid and showed tinctorial properties as keratohyalin granules show in vivo.

Alcohol oxidase and catalase in peroxisomes of methanol-grown Candida boidinii.

Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3%. As shown by electron microscopy the peroxisomes were 0.4-0.6 mum in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electrophoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria.

Gel electrophoretic studies on proteoglycans and collagen of abnormal human growth cartilage: proteoglycan abnormalities in pseudoachondroplasia and in Kniest's disease.

The microchemical study of growth cartilage biopsies may improve the classification and the genetic advice of some types of growth disturbances and contribute to the understanding of biochemical defects. Small tibial growth cartilage biopsies were performed during orthopedic surgery in cases with achondroplasia, pseudoachondroplasis (three cases), Kniest's disease (two cases), diastrophic dwarfism (two cases), parastrematic dwarfism, pycnodysos tosis, mucolipidosis type III, Blount's disease, and in three normal growing children. Five human fetal cartilages were also studied. The proteoglycans were extracted with 4 M guanidinium chloride. After dialysis against 8 M urea at pH 7, the proteoglycans were obtained by ion chromatography in urea on DEAE-cellulose and submitted to gel electrophoresis on polyacrylamide-agarose gels. The gel electrophoresis of the proteoglycans of growth cartilage of normal growing children gave two metachromatic bands situated close one to another. The proteoglycans extracted from fetal growth cartilage gave a single band with a slightly slower migration. An abnormal gel electrophoretic pattern was found in pseudoachondroplasia and in Kniest's disease. In pseudoachondroplasia a single wide band was found; in overcharged tubes several thin, more rapid bands appeared in addition to the main band. In Kniest's disease three bands were found. In all of the other syndromes studied two normally or almost normally situated bands were present. Small differences in the width and intensity of the bands observed in several cases were difficult to assess. In all cases except mucolipidosis III and Kniest's disease the collagen was extracted by using limited cleavage and solubilization with pepsin, purified and analyzed by polyacrylamide gel electrophoresis. A type of collagen with a single alpha band was found.

Enzymic activity of the second component of complement

Isolated C2 and C2i preparations were able to hydrolyze a number of synthetic esters containing basic amino acids, among which N-alpha-acetylglycyl-L-lysine methyl ester (AcGlyLysOMe) was most susceptible. The cleaving activity was a property of the C2 molecule, since it correlated with the presence of C2 on analyses of C2 preparations by ultracentrifugation in sucrose gradients, filtration through Sephadex G-200 columns, and on electrophoresis in acrylamide gels. Furthermore, acrylamide gel electrophoretic studies showed a shift in hydrolytic activity from the position occupied by C2 to that characteristic of C2i after incubation of C2 with C1s. The action was enzymatically mediated as evidenced by a bell-shaped pH activity curve, a linear dependence on C2 concentration, and the presence of Michaelis-Menten kinetics. The Michaelis constant for cleavage of AcGlyLysOMe by C2 was 1.8 X 10(-2) mol. Cleavage of C2 by C1s increased C2 enzymatic activity, yet chemical oxidation of the molecule, although enhancing hemolytic acitivity, failed to increase C2 hydrolytic activity. The observed enzymatic activity of C2 was found to be relevant to the function of C2 in the C42 complex, since AcGlyLysOMe competitively inhibited the C42 mediated cleavage of C3 in free solution and the C42 dependent binding of C3 to cells.

Regulation of oogenesis, female specific protein production, and male and female reproductive gland development by juvenile hormone in the butterfly,Nymphalis antiopa

The role of the juvenile hormone (JH) in the regulation of oogenesis and the development of the male and female reproductive glands in the butterflyNymphalis antiopa was examined by means of classical endocrine methodology. The use of neck ligatures and allatectomies, with or without injections of compounds with high JH activity, demonstrated that JH is required for: 1) oogenesis and colleterial gland development in posteclosion adult females, and 2) tubular gland-ejaculatory duct and accessory gland enlargement in posteclosion adult males. Additionally, disc gel electrophoretic studies of hemolymph from males and females in several different physiological states, and of homogenates of mature oocytes indicated that JH is required for the normal presence in gravid female hemolymph of an apparent vitellogenin.

Purification and properties of Arthrobacter ureafaciens inulase II

1. 1. Arthrobacter ureafaciens inulase II, which converts inulin to di- d-fructofuranose 1,2′:2,3′ dianhydride (difructose anhydride III) and a small amount of oligosaccharides, was purified about 150-fold in the 43% yield from the cultured liquid of the bacteria by means of ammonium sulfate fractionation, followed by acetone fractionation and Sephadex G-100 gel filtration. A polarimetric measurement was carried out for the estimation of enzymatic activities. 2. 2. The purified enzyme was found to be homogeneous by polyacrylamide disc gel electrophoretic studies in support of an intramolecular transfructosidation reaction of the enzymatic reaction. The enzyme was optimally reactive at pH 6.0 and at 50 °C, and was stable within a broad pH range (pH 4 to 11) and below 50 °C. The activity was strongly inhibited by HgCl 2.