Electrophoretic Forms Research Articles

Characterization of the altered form of the c-src gene product in neuronal cells.

The pp60c-src protein that is expressed at high levels in cultures of neurons from rat embryos displays an altered mobility on SDS-polyacrylamide gels due to a structural difference in the amino-terminal region of the molecule. In this report we show that the expression of this unique form of pp60c-src, designated pp60c-src(+), is not restricted to cultured neuronal cells since the pp60c-src molecules expressed in tissues from avian and rat neural tissues also display a retarded electrophoretic mobility. The amino-terminal region from pp60c-src(+) was found to contain a novel phosphorylated tryptic peptide that contains phosphoserine. However, this phosphorylation does not appear to be responsible for the retarded electrophoretic mobility of pp60c-src(+), since the mobility of this protein is not altered by phosphatase treatment under conditions that remove greater than 95% of the radiolabeled phosphate on pp60c-src(+). The altered electrophoretic form of pp60c-src was also shown to be radiolabeled with [3H]myristate, indicating that pp60c-src is fatty-acylated in neurons, as is pp60c-src in fibroblasts. The pp60c-src molecules synthesized in vitro using rabbit reticulocyte lysates programmed with mRNA from embryonic brain migrated more slowly on SDS-polyacrylamide gels than the pp60c-src protein that was translated in vitro using RNA from embryonic limb tissue. These results suggest the possibility that the c-src mRNA expressed in neurons may undergo a unique form of processing to generate the structurally distinct form of neuronal pp60c-src(+).

Thermal gels: A procedure for determination of heat-inactivation temperature of enzymes

A facile procedure for the study of inactivation temperatures of enzymes that could be stained for activity after electrophoresis is described. This is particularly suitable for enzymes that have multiple electrophoretic forms. The results of studies on Drosophila dehydrogenases and acetylcholinesterase are presented. The procedure may be useful in the study of genetic variants.

Developmental regulation of juvenile hormone esterase in Trichoplusia ni: Its multiple electrophoretic forms occur during each larval ecdysis

Juvenile hormone esterase activity has been found during the intramoult period of each larval stadium in Trichoplusia ni. The activity is indistinguishable from that occurring during the final larval stadium, on the basis of its four isoelectric forms and kinetic data. The 4th- to 5th-instar intramoult peak in activity also occurs in other Lepidoptera ( Heliothis virescens, Spodoptera exigua, Manduca sexta and Hyphantria cunea). Further, the final species also possessed a peak of activity during the intramoult period to the penultimate larval instar. The findings have important implications for the current concept that the function of juvenile hormone esterase is, by reason of its anti-juvenile hormone action, an enzyme of the last larval instar which enables metamorphosis to begin.

Correlation between a female sterile mutation and a set of follicle cell proteins in Drosophila melanogaster

In order to correlate the synthesis of a previously described set of follicel cell (Fc) proteins with a known mutation that affects female fertility, three female sterile mutations, fs(1)384, fs(1)508 and fs(1)1501, mapping in the same region as the Fc locus (7C1-9), were analysed with respect to Fc synthesis. The fs(1)508 strain displayed a normal Fc protein pattern, while in fs(1)384 no Fc protein synthesis could be detected. The fs(1)1501 pattern of Fc polypeptide synthesis was totally different from that of any previously analysed strain, displaying a set of proteins that were much larger than the standard Fc variant form. Two of the female sterile mutations, fs(1)384 and fs(1)1501, were combined in rans with two wild-type strains displaying two different electrophoretic variant forms of the Fc proteins. The combinations were then analysed for Fc protein synthesis, using the fact that females heterozygous for two of the Fc variant forms display both parental forms. The results indicate that the fs(1)384 mutation is directly involved in the synthesis of the Fc proteins, as the trans heterozygotes only synthesize the Fc form derived from the wild-type parent. We also suggest that the large proteins synthesized by the fs(1)1501 mutant are a defective Fc variant form. The nature of the two mutations is also discussed.

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors.

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.

Heterogeneity of Catalase in Maturing and Germinated Cotton Seeds

To investigate possible charge and size heterogeneity of catalase (EC 1.11.1.6) in cotton (Gossypium hirsutum L. cv Deltapine 62), extracts of cotyledons from different developmental ages were subjected to nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Special precautions (e.g. fresh homogenates, reducing media) were necessary to prevent artefacts due to enzyme modification during extraction and storage. When the gels were stained for enzyme activity, two distinct electrophoretic forms of catalase were resolved in extracts of maturing and mature cotton seeds. In germinated seeds, three additional cathodic forms were detected revealing a total of five electrophoretic variants. In green cotyledons, the two anodic forms characteristic of ungerminated seeds were less active; whereas, the most cathodic form was predominant. All forms of catalase were found in isolated glyoxysomes. Corresponding electrophoretic patterns were found on Western blots probed with anticatalase serum; no immunoreactive, catalytically inactive forms were detected. Western blots of sodium dodecyl sulfate-polyacrylamide gels revealed only one immunoreactive (55 kilodaltons) polypeptide in cotton extracts of all developmental ages. Results from isoelectric focusing and Ferguson plots indicate that the electrophoretic variants of catalase are charge isomers with a molecular weight of approximately 230,000.

PEROXIDASE ZYMOGRAMS OF 16 APPLE ROOTSTOCKS

Shoot bark and root extracts of 16 different apple (Malus domestica Borkh.) root-stocks were analyzed for multiple electrophoretic forms of peroxidase (EC 1.11.1.7) using polyacrylamide gel electrophoresis (PAGE). Thirteen bands were seen in the peroxidase zymograms of shoot bark extracts and eight in the peroxidase zymograms of root extracts. The root peroxidase bands corresponded to ones in the shoot bark peroxidase zymograms. Two of the bands were common to all 16 rootstock patterns. The other 11 bands formed six patterns which were characterized by the presence of specific peroxidase bands. The 16 rootstocks were grouped according to banding characteristics.Key words: Apple, Malus domestica Borkh., apple rootstocks, PAGE-peroxidase zymograms

Effect of Different Carbon Sources on the Ammonium Induction of Different Forms of NADP-Specific Glutamate Dehydrogenase in Chlorella sorokiniana Cells Cultured in the Light and Dark

The ammonium induction of the chloroplast-localized NADP-specific glutamate dehydrogenase (NADP-GDH) was shown not to be a light-dependent process per se in Chlorella sorokiniana. In the dark without exogenous organic substrates, the cells synthesized low levels of fully active NADP-GDH, provided endogenous starch reserves had not been depleted. When cells were supplied with exogenous acetate, the rate of induction of NADP-GDH activity per milliliter of culture in the dark was equal to or slightly greater than the rate observed under photosynthetic conditions without an organic carbon source. Glucose supported only a low rate of induction of NADP-GDH activity in the dark. Both acetate and glucose inhibited induction of enzyme activity in the light. The NADP-GDH holoenzyme had at least 7 different electrophoretic forms. These forms differed in net charge and/or molecular weight. Their difference in molecular weight was due to the presence of 2 subunits with similar antigenic properties but different molecular weights (M(r) = 55,500 and 53,000; alpha-and beta-subunits, respectively). Depending upon the cultural conditions and length of the induction period, a wide variation was observed in the alpha:beta subunit ratio and in the numbers and sizes of the NADP-GDH holoenzymes.

Parallel Postnatal Development of Choline Acetyltransferase Activity and Muscarinic Acetylcholine Receptors in the Rat Olfactory Bulb

The development of cholinergic synapses in the rat olfactory bulb was investigated by measuring changes in the activity of choline acetyltransferase (ChAT; EC 2.3.1.6.), a presynaptic cholinergic marker, and in the concentration of muscarinic receptors, components of cholinoceptive membranes. Three biochemical properties of the muscarinic system also were examined for possible differentiation: ligand binding, molecular weight, and isoelectric point. Receptors from embryonic (day 18), neonatal (postnatal day 3), and adult rat olfactory bulbs exhibited identical complex binding (nH = 0.45) of the agonist carbachol. For each age, the relative proportions of high-affinity (Ki approximately equal to 1.0 microM) and low-affinity (Ki approximately equal to 100 microM) binding states were 60% and 40%, respectively. The antagonist pirenzepine also bound to high-affinity (Ki approximately equal to 0.15 microM, RH approximately equal to 70%) and low-affinity (Ki approximately equal to 2.0 microM, RL approximately equal to 30%) sites in neonatal and adult rats. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis of [3H]propylbenzilylcholine mustard-labeled receptors from neonatal and adult rats showed a single electrophoretic form with an apparent molecular weight of 65,000. In contrast, analytical isoelectric focusing indicated high pI (4.50) and low pI (4.00) receptor forms were present. Neonatal rats contained approximately equal proportions of the two receptor forms, whereas adult rats contained mainly the low pI form, indicating that molecular alteration of the receptor population had occurred during development. Comparison of postnatal changes in acetylcholine receptors and ChAT activity showed a striking correlation between the development of cholinergic terminals and muscarinic receptors. Throughout the first postnatal week, ChAT activity remained at 5% of adult levels; activity began to rise on postnatal day 6 and gradually reached adult levels (56 +/- 4 mumol of [3H]acetylcholine/h/g) during the fourth week. Similarly, muscarinic receptor concentration was low (30-50 fmol/mg) throughout the first week, began to rise at postnatal day 7; and reached 90% of adult levels (317 +/- 17 fmol/mg) by the fourth week. In contrast, there was little increase in the concentration of nicotinic acetylcholine receptors (30 fmol/mg) during this period. The parallel postnatal development of ChAT activity and muscarinic receptors suggests the existence of factors that couple the differentiation of presynaptic cholinergic terminals and postsynaptic cholinoceptive elements.

Genetic analysis of subunits of two major storage proteins (.BETA.-conglycinin and glycinin) in soybean seeds.

Eight hundred and fifty soybean accessions were screened for genotypes with electrophoretic variations of the β-conglycinin and glycinin subunits. We detected two abnormal SDS-gel electrophoretic forms of β-conglycinin α-subunit, the slow electrophoretic mobility type designated as α-slow-type and the low α-subunit content type designated as α-low-type. Each of these two forms was controlled by a codominant allele at a single locus. The gene symbol Cgy2a was assigned to the common type and Cgy2b to the α-slow-type. We found a new electrophoretic form of β-conglycinin β-subunit designated as β'-subunit which migrates slightly faster than the β-subunit on SDS-gels. Beta-prime (β') subunit was controlled by a single dominant gene designated as Cgy3. The homozygous recessive cgy3 cgy3 lacked the β'-subunit. We observed two variations of the glycinin group-I acidic subunits (A1, A2 and A3) by alkaline urea electrophoresis, i. e., the concomitantly slow and fast electrophoretic mobility types designated as A(I)-slow-type and A(I)-fast-type, respectively. The concomitantly slow and fast migrating Al'A2 and A3 subunits, respectively, behaved in the F2 seeds as a single block composed 'of the three electrophoretic bands. Each of these electrophoretic forms was inherited as if controlled by a codominant allele at a single locus. The gene symbols, Gyla, Gylb and, Gylc were assigned to the alleles for the common electrophoretic type, the A(I)-slow-type and the A(I)-fast-type, respectively. Close linkage was found between Cgy2 and Cgy3. The gene Cgy1 controlling the β-conglycinin α'-subunit was independent of Gy1 and Gy4 which controls the A5 subunit and its associated basic subunit.

Each Phaseolus vulgaris isolectin exists in five electrophoretic forms related to the number of metal ions bound per molecule

Analysis of the pH 8.3 electrophoresis of the Phaseolus vulgaris seed lectins suggests that each of the five isolectins exists in five different forms. Incubations of the lectins in the presence of EDTA or of metal ions such as Mn 2+, Mg 2+, Co 2+ and Ni 2+ shows that their different electrophoretic behaviours are related to the binding of metal ions to the molecules. Determinations of the isolectin metal content by atomic absorption measurements demonstrate that at pH 8.3 their five electrophoretic forms are related to the number of subunits (0, 1, 2, 3 or 4) which contain metal ions in the tetrameric molecules.

Enrichment of dystrophy-associated antigen from erythrocyte membranes of mice with muscular dystrophy.

In a previous report, it was shown that erythrocyte membranes from mice with muscular dystrophy of strain 129/ReJ-dy manifest a unique antigen. In the current study, this antigen was enriched from solubilized membranes by a two-step solid phase immunoadsorbent. The enriched fraction retained antigenicity as well as a reduced set of electrophoretic forms of protein over that seen in solubilized membranes. The enriched fraction contained no novel molecular weight species but several were noticeably enriched, particularly at 54,000 daltons.

Comparison of antiproteolytic activities of alpha-1-proteinase inhibitors from the plasma of some mammalian species

1. 1. Alpha-1-proteinase inhibitors isolated from plasmas of horse, ox, pig, rabbit and man were used for determination of some kinetic parameters of interaction with three horse leucocyte proteinases and bovine pancreatic trypsin and chymotrypsin. 2. 2. Effective molar ratio of enzyme-to-inhibitor, inactivation rate constant and inhibition constant were measured. 3. 3. In horse, ox, pig and rabbit two principal electrophoretic forms of α 1-PI could be distinguished. 4. 4. Both forms effectively inhibited trypsin but usually only one form reacted promptly and stoichiometrically with chymotrypsin and leucocyte elastases. 5. 5. It appears that genetic variability and functional heterogeneity of multiple forms of α 1-PI as well as lack of other tissue inhibitors of proteinases may be responsible for lung emphysema occurring in man and horse.

Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system: switch in predominant form during differentiation of synapses.

Muscarinic acetylcholine receptors from the avian central nervous system were examined for developmental changes that correlated with the differentiation of cholinergic synapses. In contrast to previous studies that showed a single molecular weight form of muscarinic receptors in the mature central nervous system, the current study of receptors from embryonic and newly hatched chick retina showed the presence of two electrophoretic forms having apparent molecular weights of 86,200 +/- 400 and 72,200 +/- 300. Two receptor forms also were observed in embryonic cerebrum, optic tectum, and cerebellum. Each form was present, although decreased in molecular weight by 6000, after treatment with deglycosylating enzymes, consistent with molecular differences occurring in the protein portions, rather than the carbohydrate portions, of the molecules. The relative proportions of the high and low molecular weight receptors in retina showed a striking inversion during development. Before synaptogenesis, receptors were mainly of Mr 86,000, whereas after synaptogenesis, receptors were mainly of Mr 72,000. Development of a predominantly low molecular weight receptor population also occurred in aggregate, but not monolayer, cell culture, suggesting a possible role for cell-cell interactions in triggering the change. Pulse-chase labeling of receptors on cultured cells indicated that both forms were present on the cell surface; the labeled Mr 86,000 population had a half-life of 5 hr, whereas the labeled Mr 72,000 population had a half-life of 19 hr. The change in size of muscarinic receptors during development may reflect the action of regulatory mechanisms critical to the proper assembly and function of synapses in the central nervous system.

Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12.

Two membrane-bound hydrogenase isoenzymes present in Escherichia coli during anaerobic growth have been resolved. The isoenzymes are immunologically and electrophoretically distinct. The physically more abundant isoenzyme (hydrogenase 1) contains a subunit of Mr 64,000 and is not released from the membrane by exposure to either trypsin or pancreatin. The second isoenzyme (hydrogenase 2) apparently contributes the greater part of the membrane-bound hydrogen:benzyl viologen oxidoreductase activity and exists in two electrophoretic forms revealed by nondenaturing polyacrylamide gel analysis. This isoenzyme is irreversibly inactivated at alkaline pH and gives rise to an active, soluble derivative when the membrane-bound enzyme is exposed to either trypsin or pancreatin. Both hydrogenase isoenzymes contain nickel.

The molecular structure of different polymorphic forms of canine C3 and C4.

The subunit composition of different electrophoretic forms of canine C3 and C4 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of reduced immune precipitates. Canine C4 comprises alpha, beta, and gamma chains of approximate molecular weight 90 000-95 000, 72 000, and 33 000, respectively. The molecular weight of the alpha chain of the C4 1 allelic product was approximately 95 000, but 90 000 for the C4 2 and C4 4 allotypes. No differences were observed in the molecular weights of the beta or gamma chains of any canine C4 phenotype tested. Canine C3 appears to be encoded by a single locus. The subunit composition comprises an alpha and beta chain with molecular weights of approximately 106 000 and 71 000, respectively. Unlike C4, no differences in the molecular weights of the subunits were observed in different electrophoretic forms of canine C3.

Metabolic response to ethanol and isopropanol in D. funebris and D. immigrans

1. Ethanol at low concentration increases larval viability in D.funebris and D. melanogaster and decreases it in D. immigrans. Isopropanol decreases viability in all three species. 2. Isopropanol depresses alcohol dehydrogenase activity. This is accompanied by a change in the relative proportion of electrophoretic forms of the enzyme. 3. Acetone appears in the medium when larvae of D.funebris and D. immigrans develop in the presence of isopropanol. It can be transformed again into isopropanol on reaching a threshold value. It seems that this transformation cannot be carried out by alcohol dehydrogenase alone.

Phaseolus vulgaris lectin heterogeneity related to their metal content

The seed lectins of Phaseolus vulgaris cv. Contender typically consist of five tetrameric isolectins E4, E3L, E2L2, EL3 and L4. However electrophoretic and isoelectric focusing analysis of each purified protomer show a great heterogeneity. We demonstrate that each tetrameric isolectin exists in five different electrophoretic forms depending on the amount of Mn2+ cation bound to each isolectin. The effects of Mn2+ binding on charge and/or molecular properties of the protomers are discussed. Under the same experimental conditions, if we use Ca2+ ions instead of Mn2+, the abovemodifications are not observed.

Novel carbonic anhydrase (Car-2) allele in Spanish mice.

A unique electrophoretic form of carbonic anhydrase is characteristic of some laboratory-maintained mice of the wild mouse species Mus spretus. This isozyme has been characterized by cellulose acetate electrophoresis and by isoelectric focusing. It is proposed that this isozyme be called CAR-2C and that its encoding allele be designated Car-2c. Fertile hybrids of Mus spretus and C57BL/6J (Car-2a) show both CAR-2A and CAR-2C bands of approximately equal intensity. The CAR-2C isozyme is readily identified by electrophoresis on 75-mm cellulose acetate strips because it migrates significantly faster than the isozymes of inbred mice, the CAR-2A and CAR-2B that do not separate from one another under standard conditions. Isoelectric focusing cleanly resolves all three of these CAR-2 forms. Mus hortulanus, although closely related to Mus spretus in other biochemical-genetic characteristics, has a CAR-2-homologous isozyme that is distinctly different from the CAR-2C of Mus spretus and from the isozymes of the common inbred strains.

Alkaline phosphatase isozymes in large intestines and large intestinal tumors of Fischer 344 rats

Eight electrophoretic forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) were detected in butanol extracts of Fischer 344 rat large intestines. Seven of these isozymes have higher mobility than the small intestinal form(s) in non-denaturing polyacrylamide gel electrophoresis. These more mobile forms may be derived during extraction from more slowly migrating forms that are analogous to those forms found in the small intestine. Monoclonal antibody with specificity for a rat small intestinal isozyme cross-reacts with four of the large intestinal isozymes. This antibody does not cross-react with alkaline phosphatases from human or hog small intestine. Gel exclusion chromatography molecular weight estimates of rat intestinal forms range from 1.1 X 10(5) to 2.5 X 10(5). Alkaline phosphatase from two colon tumors obtained from azoxymethane treated rats appeared to be similar to an isozyme found in some normal rats, on the basis of electrophoretic mobility and cross-reactivity with the monoclonal antibody.