Polyacrylamide Gel System Research Articles

Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.

Heat shock led to marked changes in the apparent levels of phosphorylation of nuclear proteins in the fungus Achlya ambisexualis. We characterized these heat shock-induced changes in nuclear proteins on two types of two-dimensional polyacrylamide gel systems. We report here that one of two Achlya H3 histones (H3.1) and also the oomycete histone alpha appear to be highly phosphorylated with heat shock. Additional changes observed in acid-soluble nuclear proteins included an apparent increase in the 32P labeling of a 43,000-molecular-weight protein and the dephosphorylation of a major group of Achlya phosphoproteins in the 30,000-to-32,000-molecular-weight range. The changes in protein phosphorylation were accompanied by striking changes in the morphology of Achlya nuclei. Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells.

Hydrophobic and ionic effects upon the electrophoretic mobilities of the subunits of coupling factor 1 from mitochondria

A sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system was used to investigate certain properties of the subunits of the beef heart mitochondrial ATPase, (native F1, nF1). By examining the affects of urea concentration and acrylamide concentration upon the electrophoretic mobilities of the polypeptides comprising the nF1 enzyme, we have obtained conditions under which all five subunits are simultaneously resolved when the discontinuous buffer system of Laemmli is used ( U. K. Laemmli (1970) Nature (London) 277, 680–685). The determination of the apparent molecular weights by analysis of Ferguson plots ( K. A. Ferguson (1964) Metabolism 13, 985–1002) revealed that the addition of urea to the SDS gels resulted in a decrease in the apparent molecular weight of the β subunit. A dramatic increase in the apparent molecular weight of the δ subunit was also brought about by the presence of urea in the SDS gels. In addition, the apparent molecular weight of both the α and the β subunits was dependent upon the acrylamide concentration used, indicating that these subunits contain either areas highly resistant to denaturation by the combined action of urea and SDS, or covalent modifications leading to anomalous electrophoretic mobility. The results of experiments in which urea analogs were used indicate that the interactions of urea with the β subunit involve the formation of hydrogen bonds between urea and regions of this subunit. On the other hand, the interactions of urea with the δ subunit are primarily of a hydrophobic nature, suggesting that these interactions could involve domains of the δ subunit required for binding of the coupling factor to the mitochondrial membrane.

Characterization of thrombin binding to alpha 2-macroglobulin.

The formation and structural characteristics of the human alpha 2-macroglobulin (alpha 2M)-thrombin complex were studied by intrinsic protein fluorescence, sulfhydryl group titration, electrophoresis in denaturing and nondenaturing polyacrylamide gel systems, and in macromolecular inhibitor assays. The interaction between alpha 2M and thrombin was also assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digests of denatured alpha 2M-125I-thrombin and alpha 2M-125I-trypsin complexes. In experiments measuring fluorescence changes and sulfhydryl group exposure caused by methylamine, we found that thrombin produced its maximum effects at a mole ratio of approximately 1.3:1 (thrombin:alpha 2M). Measurements of the ability of alpha 2M to bind trypsin after prior reaction with thrombin indicated that thrombin binds rapidly at one site on alpha 2M, but occupies the second site with some difficulty. Intrinsic fluorescence studies of trypsin binding to alpha 2M at pH 5.0, 6.5, and 8.0 not only revealed striking differences in trypsin's behavior over this pH range, but also some similarities between the behavior of thrombin and trypsin not heretofore recognized. Structural studies, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure alpha 2M-125I-thrombin covalent complex formation, indicated that covalency reached a maximum at a mole ratio of approximately 1.5:1. At this ratio, only 1 mol of thrombin is bound covalently per mol of alpha 2M. These gel studies and those of proteolytic digests of denatured alpha 2M-125I-trypsin and alpha 2M-125I-thrombin complexes suggest that proteinases form covalent bonds with uncleaved alpha 2M subunits. The sum of our results is consistent with a mechanism of proteinase binding to alpha 2M in which the affinity of the proteinase for alpha 2M during an initial reversible interaction determines its binding ratio to the inhibitor.

Use of benzyldimethyl- n-hexadecylammonium chloride (“16-BAC”), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells

A discontinuous polyacrylamide gel system operating at pH 4.0–1.5 which resolves proteins bearing base labile groups extracted from intact cells is described. It uses potassium phosphate buffer in the running and stacking gel and glycine as the trailing ion component. Proteins are solubilized with urea and benzyldimethyl- n-hexadecylammonium chloride, a cationic detergent. The utility of the system is illustrated by fluorographs of the pattern of protein methylation in blood platelets and the HL60 promyelocyte cell line.

Assignment of a polymorphic polypeptide to the human mitochondrial DNA unidentified reading frame 3 gene by a new peptide mapping strategy.

A new method of peptide analysis is presented which allows assignment of unknown proteins to coding regions of genomes which have been sequenced. This approach involves comparison of the molecular weights of peptides generated by partial proteolytic digestion with those predicted for a protein whose primary amino acid sequence is deduced from a corresponding nucleotide sequence. The proteolytic digestions are accomplished in situ in the stacking gel of a two-dimensional polyacrylamide gel system. We have used this system to show that two variant proteins of the human mitochondrial DNA, MV-1 and MV-2, are allelic and encoded by the unidentified reading frame 3 (URF 3) gene. This assignment was supported by sequence analysis of a clone of this mtDNA region from a HeLa cell line which expresses the uncommon variant MV-2. Four nucleotide changes were found in HeLa URF 3, relative to the reported sequence from human placenta. Two of these changes alter the primary amino acid sequence of the encoded protein. It is proposed that one of those amino acid changes may account for the observed molecular weight variation in MV-1 and MV-2 by proteolytic cleavage, conformational change, or secondary modification. We have used this method to also assign a mitochondrially translated protein to URF 6. These are the first assignments of mitochondrially synthesized polypeptides to human URF genes and prove conclusively that at least some of these genes are expressed in human cells.

The seed proteins of chick pea: Comparative studies ofCicer arietinum, C. reticulatum andC. echinospermum (Leguminosae)

Seed proteins fromCicer arietinum L.,C. reticulatumLadiz. andC. echinospermumDavis were extracted and separated into water soluble (albumin) and water insoluble (globulin) fractions. These were analysed using three polyacrylamide gel systems: uniform pore slab gels, gradient gels and SDS disc gels. For all three species, albumins constitute just over one-third of total protein. Minor differences in the composition of this fraction were observed. Within the globulin fraction, seven disulphide-linked polypeptides were found. Four of these resemble the major polypeptide of legumin, consisting of constant small subunit (21,000 daltons) linked to variable large subunit (46,000, 41,000, 39,000 or 36,000 daltons), forming polypeptides of 67,000 (I), 62,000 (II), 60,000 (III) and 57,000 (IV) daltons respectively. Polypeptide I was prominent in both wild species, but absent fromC. arietinum. Polypeptides II and III were equally prominent inC. arietinum andC. reticulatum. Polypeptide IV was more prominent inC. echinospermum, which was deficient in polypeptide III. Polypeptides V (45,000 daltons) and VI (43,000 daltons), apparently composed of two equal subunits, were present in trace amounts in both wild species, but well represented inC. arietinum Polypeptide VII of 45,000 daltons (31,000 + 14,000) was present in all three species.

4] Measurement of hormone-stimulated protein phosphorylation in intact cells

This chapter discusses the measurement of hormone-stimulated protein phosphorylation in intact cells. The use of radioactive phosphate to measure the phosphorylation state of enzymes in intact cells began in 1956, using the approach to demonstrate that epinephrine and glucagon stimulated the phosphorylation of phosphorylase in dog liver slices. The recent development of sodium dodecyl sulfate (SDS)-polyacrylamide gel systems capable of resolving very large numbers of proteins has increased the utility of this technique and it is now being applied to a number of tissues responsive to hormones or neurotransmitters. The advantages of measuring changes in protein phosphorylation in intact cells are (1) the protein kinases and substrates are in their native state during the phosphorylation reaction, obviating arguments about the relevance of the phosphorylation events and (2) changes can be observed in the phosphorylation states of proteins when the hormone message or the protein kinase mediating the reaction are not clearly defined.

Comparison of low- and high-calcium-requiring forms of the calcium-activated protease with their autocatalytic breakdown products

The effect of controlled autocatalytic degradation on the subunit molecular weight, subunit composition and calcium sensitivity of the low-calcium-requiring and the high-calcium-requiring forms of the calcium-activated muscle protease was examined. Purified low- and high-calcium-requiring proteases coelectrophorese on SDS-polyacrylamide gels. Controlled autocatalysis of either form of the protease results in extensive degradation of their respective 30 kDa subunits and partial degradation of their 80 kDa subunits. In this electrophoresis system the electrophoretic banding pattern of the low-calcium-requiring protease is clearly different from that of the autocatalytically degraded high-calcium-requiring protease. Similar results were obtained using a nondenaturing polyacrylamide gel system. Both the high- and low-calcium-requiring proteases were made more sensitive to calcium by autocatalytic degradation. However, the results of this study strongly indicate that autocatalytic degradation does not resultin conversion of high-calcium-requiring protease to the low-calcium-requiring protease as has been recently hypothesized (Suzuki et al. (1981) J. Biochem. 90, 275–278).

Digestive proteinases of Sitophilus weevils (Coleoptera: Curculionidae) and their response to inhibitors from wheat and corn flour

Hydrolysis of two general proteinase substrates and seven substrates specific for trypsin-like and chymotrypsin-like enzymes, aminopeptidases, and carboxypeptidases was demonstrated by homogenates of alimentary systems of larvae and adults of Sitophilus oryzae (L.), S. zeamais Motchulsky, and S. granarius (L.). Endopeptidase activity was very low in all species, whereas aminopeptidases and carboxypeptidases were relatively active. Hydrolysis of α-N-benzoyl-DL-arginine-p-nitroanilide (BApNA) by larvae and adults of S. granarius was due to a single enzyme, inhibited by N-α-tosyl-L-lysinechloromethyl ketone (TLCK) and soybean trypsin inhibitor, and with a relative mobility of 0.49 on an anionic polyacrylamide gel system. This enzyme was not detected in S. oryzae or S. zeamais. Aminopeptidases (hydrolysis of L-leucine-p-nitroanilide) were inhibited by 1, 10-phenanthroline, Cd2+, and Hg2+, and existed in two major and two to three minor isozymes in each species. Water extracts of wheat and corn flour inhibited the BApNA enzyme from S. granarius but had no effect upon the aminopeptidases and carboxypeptidases (hydrolysis of N-carbobenzoxy-L-valyl-L-leucine).

In Vitro cotranslational processing of human pre-interferon β1 enhances its biological activity

The addition of dog pancreas microsomes to a micrococcal nuclease-treated rabbit reticulocyte system during cell-free translation of Hu IFN- β 1 mRNA resulted in a 5- to 33-fold increase in the yield of antiviral activity per microgram of mRNA. Size analysis of the immunoprecipitated translation products showed that this increase in biological activity was concomitant with the conversion of an 18,000-dalton species (preIFN) into two products of 16,500 and 20,000 daltons, respectively. In the polyacrylamide gel system used, the peak of the latter product coincided with that of authentic Hu IFN- β 1. This method of translation has been applied to the rapid scanning of IFN- β 1 mRNA activity after sucrose gradient purification and to the quantitative analysis of IFN- β 1 mRNA.

Equimolar heterodimers in microtubules.

Two equimolar beta chains can be resolved from sea urchin sperm flagellar and scallop gill ciliary tubulins, and from certain brain tubulins as well, using the Triton X-100-acid-urea polyacrylamide gel system commonly used for histone analysis. The beta chains are identified as such from their mobility on urea-free SDS PAGE, from amino acid composition, and from tryptic peptide distribution. Scallop beta chains have almost identical amino acid profiles but they differ by one tryptic peptide. Optimal conditions for beta chain resolution are very species-dependent, with some closely related species showing either maximal or no beta chain separation. In addition, beef brain tubulin on Triton X-100-acid-urea electrophoresis and scallop gill ciliary tubulin upon isoelectric focusing in the presence of SDS show two approximately equimolar alpha chains. These data, indicating equimolar amounts of two potentially different tubulin heterodimers from a variety of microtubule types, support a model for microtubule structure wherein protofilaments consist of alternating heterodimers of two kinds, generating a 16-nm (2-dimer) axial repeat.

Turnover of mitochondrial inner membrane proteins in hepatoma monolayer cultures.

The degradation rates of inner mitochondrial membrane proteins were examined in serum-deprived hepatoma cultures. In those nonproliferating cells, the degradation of composite mitochondrial proteins was a first order process with a half-life of 34 h. The half-lives of specific inner mitochondrial membrane polypeptides were determined by examining the 3H/35S of isolated polypeptides from cells given [3H]methionine and [35S]methionine pulses, respectively, before and after a 2-day chase period. The 33 most abundant polypeptides resolved on a bidirectional polyacrylamide gel system showed half-lives ranging from 20 to 100+ h. The 15 polypeptides translated on mitochondrial ribosomes in the presence of inhibitory concentrations of cycloheximide also displayed heterogeneous rates of degradation (t1/2 = 35-100+ h). None of the isolated adenosine triphosphatase (coupling factor F1) or immunoprecipitated cytochrome c oxidase subunits were significantly turned over during the case period. Five of eight cytochrome b-c1 complex subunits, however, were turned over significantly more rapidly (t1/2 = 39-42 h) than the other three (t1/2 = 94+ h). The results demonstrate heterogeneous degradation rates for inner membrane polypeptides, extending in some cases to those within the same respiratory complex.

Type V Collagens of the Human Placenta: Trimer α-Chain Composition, Ultrastructural Morphology and Peptide Analysis

The α-chain trimer composition of type V collagen preparations from human placental membrane and villi was determined by two-dimensional electrophoresis on a non-denaturing polyacrylamide gel system followed by electrophoresis in the presence of sodium dodecylsulfate. In preparations isolated from placental membranes pure type V collagen was found with the α-chain composition [α1(V)]2 α2(V). In preparations from placental villi two different collagen trimers could be identified with α-chain compositions [α1(V)]2 α2(V) and [α3(V)]3. Two-dimensional peptide maps after chymotryptic digestion of the various α-chain revealed distinct patterns for α1(V), α2(V) and α3(V) suggesting unique structures for all three α-chains. Shadowing of the two collagen preparations with carbon-platinum by the rotary shadowing technique allowed the visualization of individual molecules. In the placental membrane preparations, a uniform species of molecules was present while in placental villi preparations the same elongated form of collagen was found together with larger aggregates presumably containing molecules with the α3-chain component. These data are interpreted to indicate that so-called type V collagen, at least in preparations from placental villi, contain two distinct collagen molecules.

Acid phosphatase isoenzymes of Chlamydomonas reinhardii.

Acid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wild-type and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are related to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P2 and their total absence in mutant Pa. The pI values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either alpha-naphthyl phosphate or different acetate esters as substrates.

Effect of gonadal hormones on the incorporation of [ 3H]-lysine into proteins of human fetal brain

Samples were obtained from the hypothalamus, temporal lobe and frontal cortex of brains from human fetuses aged 14–22 weeks. Slices were prepared and incubated with estradiol, testosterone or dihydrotestosterone, and the incorporation of [ 3H]lysine into protein was compared with that of untreated control tissue. Cytosol and crude nuclear subcellular fractions from each area were made and resolution of specific proteins was carried out using a polyacrylamide gel system. Steroid hormones had no detectable effect on total protein synthesis, but incorporation of [ 3H]lysine into certain low molecular weight proteins of the hypothalamus increased significantly in the presence of estradiol and testosterone. Dihydrotestosterone was inactive. The temporal lobe and frontal cortex were unaffected by the steroid hormones.

Quantitative analysis of the protein composition of influenza A and B viruses using high resolution SDS polyacrylamide gels

The structural polypeptide composition of a number of influenza A and B viruses was analysed using a high resolution SDS polyacrylamide gel (SDS PAGE) system. The haemagglutinin (HA) content of six influenza A viruses of three antigenic subtypes was relatively constant with a mean of 34·7 ± 3·4%. A similar quantitation was observed for the HA of influenza A recombinant viruses (31·9 ± 4·1%) and influenza B viruses (32·8 ± 3·3%). The percentage content of matrix protein (M), nucleoprotein (NP) and neuraminidase and polymerase polypeptides (NA + P) in the virion was 33·5 ± 6·7, 22·6 ± 4·5 and 7·3 ± 2·3 respectively. The SDS PAGE technique appears to provide a reproducible quantitative assay of the relative proportions of the structural proteins in influenza viruses used as standard preparations for potency estimations of inactivated influenza vaccines.

The ultrasensitive silver “protein” stain also detects nanograms of nucleic acids

The ultrasensitive silver staining procedure developed for proteins also stains nanogram quantities of RNA and DNA in polyacrylamide gels. A gradient polyacrylamide gel system is described which separates proteins from 10 4 to 10 6 M r, RNA from 5S to 23S and DNA from 0.4 to 21 Kb. The sensitivity of nucleic acid silver staining in this gel system considerably exceeds that of commonly used DNA and RNA dye-binding stains.

In vitro uptake and processing of prezein and other maize preproteins by maize membranes.

A cell-free, mRNA-dependent system has been developed for the translation and processing of zein preproteins. A rough endoplasmic reticulum (RER)-enriched fraction, isolated by sucrose density gradients, can be treated with micrococcal nuclease to destroy endogenous messages. When these membranes are added to a wheat germ protein-synthesizing system together with zein mRNA, synthesis and processing of the polypeptides to the mature products takes place. The RER fraction from the endosperm has a different protein composition than that prepared from either the shoot or nucellar tissue and processes prezein more efficiently. The cleavage of the preproteins appears to be a cotranslational step as the completed preprotein chains cannot be processed, although they can be taken up to a limited extent. This small uptake, or absorption, or unprocessed zein seems to be an artifact and may be related to the unusual solubility properties of zein. Finally a sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system has been developed which is particularly suited for the separation of low molecular weight proteins (less than 10,000 daltons). Using this method, we examined the products of in vitro zein processing and detected no presequence polypeptides. This suggests that the zein cleavage proteinase is probably an exopeptidase.

Isolation and properties of digestive carboxypeptidases from midguts of larvae of the black carpet beetle Attagenus megatoma

Two cationic carboxypeptidase fractions (CMS-G-1 and CMS-G-2) were isolated from the midgut homogenate of larvae of the black carpet beetle, Attagenus megatoma, through a series of ion exchange and affinity gel chromatographic steps. Fraction CMS-G-2 was highly active against the substrates hippuryl- dl-β-phenyllactate, N- carbobenzoxy-glycyl- l-phenylalanine , and N- carbobenzoxy-glycyl- l-tyrosine and was thus similar in specificity to bovine carboxypeptidase A. Fraction CMS-G-1 was most active against N- carbobenzoxy- l-valyl- l-leucine and N- carbobenzoxy-glycyl- l-methionine . Fraction CMS-G-1 also hydrolysed (in decreasing order) blocked peptides with carboxyl terminal l-isoleucine, l-leucine, l-alanine, and l-valine but was only slightly active against the ester and peptide substrates of carboxypeptidase A. Neither CMS-G-1 nor CMS-G-2 hydrolysed N- carbobenzoxy-glycyl- l-proline or glycyl-glycine. Between CMS-G-1 and CMS-G-2, at least four bands with peptidase activity were resolved on a cationic polyacrylamide gel system. The terminal and penultimate amino acids of the peptides were equally important in determining the specificity of individual peptidase bands. Fractions CMS-G-1 and CMS-G-2 each had a single, separate activity band with the substrate N- carbobenzoxy-glycyl- l-leucine . These two peptidases had molecular weights of about 35,500 and were most active at pH 7.5 to 8.5. Both enzymes were inhibited by the metal chelators EDTA and 1,10-phenanthroline and also by heavy metals such as Cu 2+, Hg 2+ and Cd 2+. The two enzymes thus appear to be metalloproteins with active sulphydryl groups.

A modification of the acetic acid-urea system for use in microslab polyacrylamide gel electrophoresis

A modification of the S. Panyim and R. Chalkley (1969, Arch. Biochem. Biophys. 130, 337) acetic acid-urea polyacrylamide gel system to include a stacking gel has been devised. The modification makes possible the use of acetic acid-urea gels in the microslab system of P. T. Matsudaira and D. R. Burgess (1978, Anal. Biochem. 87, 386).