Isoelectric focusing of multiple forms of DNase in thin layers of polyacrylamide gel and detection of enzymatic activity with a zymogram method following separation

Multiple forms of bovine pancreatic DNase (DNases A, B, C, and D) are separated by isoelectric focusing in thin layers of polyacrylamide gel with a carrier ampholyte in the pH range 4–6. The isoelectric points of DNases A, B, C, and D are 5.22, 4.96, 5.06, and 4.78, respectively. A zymogram method for detecting DNase activity as bands in the gel following isoelectric focusing is described. The method detects microgram amounts of DNase and has only one step. It can be used with the parified cazyme as well as with crude extracts of tissues containing DNase. By this method, two major components of DNase in ovine pancreas and at least three in malted barley as well as two previously unideatified forms of DNase in bovine pancreas with isoelectric points of 5.12 and 5.48 (DNases E and F) are observed.     

The immunological and structural comparisons of deoxyribonucleases I. Glycosylation differences between bovine pancreatic and parotid deoxyribonucleases

A rabbit antiserum against bovine pancreatic DNase A is used to study the immunological reaction of DNases I. As shown by double immunodiffusion, bovine pancreatic DNases A, B, C, and D are immunologically identical, so are DNases from bovine pancreas and parotid and from ovine pancreas. These DNases also behave similarly in immunotitration of DNase activity and all are tightly bound to the immunoaffinity medium, requiring an acidic buffer with 10% ammonium sulfate to dissociate. On the other hand, porcine pancreatic and malted barley DNases that do not form precipitin lines remain active in solution with the antibody; however, in spite of the lack of inhibition these DNases are retarded (but not tightly bound) in immunoaffinity chromatography, suggesting interaction with the antibody. In thin layer isoelectric focusing, the parotid DNase, purified with the immunoaffinity technique, shows only two major active components whose isoelectric points correspond to those of DNases A and C of bovine pancreas. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of parotid DNase is 34,000, approximately 3,000 more than that of the pancreatic enzyme. However, both parotid and pancreatic DNases have the same NH2-terminal leucine, an identical COOH-terminal amino acid sequence, nearly identical amino acid compositions, and almost the same peptide maps. The molecular weight difference is due to differences in the carbohydrate side chains. Results of peptide analyses indicate that parotid DNase contains two glycopeptides; pancreatic DNase has only one. In addition, both parotid glycopeptides contain glucosamine and galactosamine while the pancreatic glycopeptide has only glucosamine.     

Purification and properties of human pancreatic deoxyribonuclease I.

Human pancreatic DNase I was purified extensively from duodenal juice of healthy subjects by a procedure including ammonium sulfate fractionation, ethanol fractionation, phosphocellulose fractionation, isoelectric focusing, and gel filtration. The final preparation was free of DNase II, pancreatic RNase, alkaline phosphatase, and protease. The enzyme had a molecular weight of approximately 30,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 7.2-7.6. It required divalent cations for activity, and caused single-strand breaks by endonucleolytic attack on double- as well as single-stranded DNA molecules. The enzyme was inhibited by actin and bovine pancreatic DNase I antibody.     

One cell–one immunoglobin. Origin of limited heterogeneity of myeloma proteins

Plasma-cell tumour 5563 forms a single molecular species of immunoglobulin IgG(2)a, i.e. one variant of heavy chain and one variant of light chain. The molecules formed are labile and undergo alterations in charge properties, which rapidly lead to heterogeneity of the myeloma protein after synthesis. The single immunoglobulin species originally formed is found only after the shortest time-intervals tested, i.e. 10min incubation. Two types of changes in charge properties take place: (1) The originally formed protein (component o) is converted via an intermediate o' into the most basic form of 5563 myeloma protein found in serum (component a). Charge differences between these components are suppressed at pH8.9, but can be studied by chromatography at pH6.5 or by analysis of isoelectric points by isoelectric focusing in polyacrylamide gel. The conversion of components o and o' into component a apparently commences soon after assembly of the molecules and proceeds to completion extracellularly. (2) The second type of charge difference that distinguishes components a, b, c and d is exhibited over the pH range 6.0-8.9, but not at acid pH, and has been studied by electrophoresis at pH8.9, by chromatography and by isoelectric focusing. Conversion of component a into components b, c, d and e is only partial so that all five components can be found at decreasing concentrations in serum. Both types of charge alteration can be effected in vitro in the presence of serum, with optimum pH8.0. None of the charge differences could be attributed to the secretion process, since a component with the same isoelectric point as component o was found in secreted myeloma protein (1h). We have found no evidence to support the idea that the first type of change from component o to component a is due to ring formation of N-terminal [(14)C]glutamine into pyrrolid-2-one-5-carboxylic acid; however, our findings do not exclude this process happening very rapidly to a precursor of component o, possibly the polypeptide chain during or immediately after synthesis. In studying this point we noted that not only the heavy chains but also the kappa-type light chain of mouse 5563 myeloma protein have a blocked N-terminus.     

Carp liver actin: isolation, polymerization and interaction with deoxyribonuclease I (DNase I).

The aim of this study was to isolate and to characterize actin from the carp liver cytosol and to examine its ability to polymerize and interact with bovine pancreatic DNase I. Carp liver actin was isolated by ion-exchange chromatography, followed by gel filtration and a polymerization/depolymerization cycle or by affinity chromatography using DNase I immobilized to agarose. The purified carp liver actin was a cytoplasmic beta-actin isoform as verified by immunoblotting using isotype specific antibodies. Its isoelectric point (pI) was slightly higher than the pI of rabbit skeletal muscle alpha-actin. Polymerization of purified carp liver actin by 2 mM MgCl(2) or CaCl(2) was only obtained after addition of phalloidin or in the presence of 1 M potassium phosphate. Carp liver actin interacted with DNase I leading to the formation of a stable complex with concomitant inhibition of the DNA degrading activity of DNase I and its ability to polymerize. The estimated binding constant (K(b)) of carp liver actin to DNase I was calculated to be 1.85x10(8) M(-1) which is about 5-fold lower than the affinity of rabbit skeletal muscle alpha-actin to DNase I.     

Human urine DNase I: immunological identity with human pancreatic DNase I, and enzymic and proteochemical properties of the enzyme

DNase I in human urine was purified to an electrophoretically homogeneous state by column chromatographies on DEAE-lignocellulose, hydroxyapatite, DEAE-cellulose, Sephadex G-75 and elastin-celite. The purified enzyme was immunologically identical with human pancreatic DNase I, but not with bovine pancreatic DNase I. The molecular weight and isoelectric point of the enzyme were estimated to be 4.1 X 10(4) and 3.6, respectively. The amino acid analysis revealed that 1 mol of the enzyme contained 8 mol of half-cystine. The N-terminal amino acid was identified as leucine by the dansyl chloride method. The enzyme was active in the presence of Mg2+, Co2+, or Mn2+, The optimum pH was around 6.5. The enzyme was stable in the pH range from 5.0 to 9.0 and at temperatures lower than 45 degrees C. The rate of hydrolysis of native DNA by the enzyme was twice as fast as that observed with heat-denatured DNA. This enzyme exhaustively degraded about 20% of the phosphodiester bonds in native DNA. The enzyme also degraded poly(dA) and poly(dT), but hardly degraded poly(dG) and poly(dC).     

Genetic polymorphism of urine deoxyribonuclease I isomerases of subterranean mole rats,Spalax ehrenbergi superspecies,in Israel: Ecogeographical patterns and correlates

Genetic polymorphism of urine deoxyribonuclease I (DNase I) of mole rats was analyzed by isoelectric focusing in a thin-layer polyacrylamide gel (IEF-PAGE). One hundred and three subterranean mole rats, comprising 13 populations belonging to the four chromosomal species (2n = 52, 54, 58, 60) of the actively speciating Spalax ehrenbergi superspecies in Israel, were tested. The following results were indicated. (i) Spalax DNase I consisted of 6-12 major isozymes. (ii) Four phenotypes (numbers in parentheses) were 1 (92), 1-2 (5), 1-3 (4), and 2 (1). The decreasing order of genetic diversity, He, in the four species was 0.37, 0.13, 0.10, and 0.0 for 2n = 58, 52, 54, and 60, respectively. (iii) Spearman rank correlations and multiple regression analyses indicated associations of allele frequencies and genetic diversity with climatic and vegetation factors. We concluded that (a) climatic selection, either directly or indirectly through plant (i.e., food resources) diversity, plays an important role in DNase genetic differentiation and (b) no gene flow and introgression occur between the recent derivative of speciation (2n = 60) and its ancestor (2n = 58), suggesting the operation of reproductive isolation between both species despite natural hybridization.     

Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution.

The shape of an actin subunit has been derived from an improved 6 A map of the complex of rabbit skeletal muscle actin and bovine pancreatic DNase I obtained by X-ray crystallographic methods. The three-dimensional structure of DNase I determined independently at 2.5 A resolution was compared with the DNase I electron density in the actin:DNase map. The two structures are very similar at 6 A resolution thus leading to an unambiguous identification of actin as well as DNase I electron density. Furthermore the correct hand of the actin structure is determined from the DNase I atomic structure. The resolution of the actin structure was extended to 4.5 A by using a single heavy-atom derivative and the knowledge of the atomic coordinates of DNase I. The dimensions of an actin subunit are 67 A X 40 A X 37 A. It consists of a small and a large domain, the small domain containing the N terminus. Actin is an alpha,beta-protein with a beta-pleated sheet in each domain. These sheets are surrounded by several alpha-helices, comprising at least 40% of the structure. The phosphate peak of the adenine nucleotide is located between the two domains. The complex of actin and DNase I as found in solution (i.e., the actin:DNase I contacts which do not depend on crystal packing) was deduced from a comparison of monoclinic with orthorhombic crystals. Residues 44-46, 51, 52, 60-62 of DNase I are close to a loop region in the small domain of actin. At a distance of approximately 15 A there is a second contact in the large domain in which Glu13 of DNase I is involved. A possible binding region for myosin is discussed.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.     

Comparison of extracellular protein profiles of seven serotypes of mutans streptococci grown under controlled conditions

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.     

Cyclic nucleotide phosphodiesterase in rat neostriatum: multiple isoelectric forms with similar kinetic properties

Separation of multiple forms of cyclic nucleotide phosphodiesterase from the soluble supernatant fraction of rat neostriatum by isoelectric focusing yielded five separate peaks of cyclic nucleotide hydrolysing activity. Each separated enzyme form displayed a complex kinetic pattern for the hydrolysis of both cyclic AMP and cyclic GMP, and there were two apparent Km's for each nucleotide. At 1 microM substrate concentration, four enzyme forms exhibited higher activity with cyclic AMP than with cyclic GMP, while one form yielded higher activity with cyclic GMP than with cyclic AMP. Cyclic AMP and cyclic GMP were both capable of almost complete inhibition of the hydrolysis of the other nucleotide in all the peaks separated by isoelectric focusing; the IC50's for this interaction correlated well with the relative rates of hydrolysis of each nucleotide in each peak. The ratio of activity at 1 microM substrate concentration for the five enzyme forms separated by isoelectric focusing was 10:10:5:15:1 for cyclic AMP hydrolysis; and 6:6:4:8:2 for cyclic GMP hydrolysis; and the isoelectric points of the five peaks were 4.3, 4.45, 4.7, 4.85, and 5.5, respectively. Known phosphodiesterase inhibitors did not preferentially inhibit any of the separated forms of activity for either cyclic AMP or cyclic GMP hydrolysis, at either high (100 microM) or low (1 microM) substrate concentrations. Preliminary examination of the subcellular distribution of the different forms of enzyme activity indicated a different degree of attachment of the various forms to particulate tissue components. Isoelectric focusing of the soluble supernatant of rat cerebellum gave rise to a slightly different pattern of isoelectric forms from the neostriatum, indicating a different cellular distribution of the isoelectric forms of PDE in rat brain. Polyacrylamide disc gel electrophoresis of the soluble supernatant of rat neostriatum also generated a characteristic pattern of five separate peaks of cyclic nucleotide phosphodiesterase activity, each of which hydrolysed both cyclic AMP and cyclic GMP. Polyacrylamide gel electrophoresis of single enzyme forms previously separated by isoelectric focusing gave single peaks, with a marked correspondence between the enzyme forms produced by isoelectric focusing and those produced by gel electrophoresis, suggesting that both protein separation procedures were isolating the same enzyme forms. The results indicate the existence of multiple isoelectric forms of cyclic nucleotide phosphodiesterase in the soluble supernatant fraction of rat neostriatum, all of which exhibit similar properties. In this tissue a single kinetic form of this enzyme appears to exist displaying complex kinetic behaviour indicative of negative cooperativity and hydrolysing both cyclic AMP and cyclic GMP, with varying affinities.     

Isoelectric focusing of interacting systems. I. Carrier ampholyte-induced macromolecular isomerization.

A phenomenological theory of isoelectric focusing is formulated for rapidly reversible, ampholyte-induced macromolecular isomerization. The calculations reveal that such interactions can give well resolved, bimodal transient and equilibrium isoelectric focusing patterns in which the two peaks correspond to different chemical equilibrium compositions and not to separated isomers. The kinetics of approach to the equilibrium pattern are characteristically biphasic: During the first phase, which is controlled by the rate of migration of the isomers in the electric field, two peaks are positioned in the region between the isoelectric points of the two isomers; one of the peaks then grows slowly at the expense of the other with a diffusion-dominated rate. The kinetics are dependent upon the initial distribution of macromolecule in the isoelectric focusing column, and in certain cases only a single peak is apparent during the first phase. These findings have practical implications for unambiguous interpretation of isoelectric focusing patterns, furnish explanations for hitherto puzzling experimental observations, and provide theoretical insights required for application of isoelectric focusing to the detection and characterization of macromolecular interactions in general.     

Geographical north-south decline in DNASE1*2 in Japanese populations

Allele frequencies for human deoxyribonuclease I (DNase I) phenotypes were determined using blood samples from about 2000 Japanese subjects living in nine prefectures, and compared with one another. DNase I phenotyping was performed principally using isoelectric focusing electrophoresis and activity staining. The DNase I system was shown to have enhanced potential for anthropologic, genetic, and clinical studies of Japanese populations. DNase I phenotypes were analyzed to evaluate the degree of genetic variation at the DNASE1 locus. Our examination of DNase I types revealed a decreasing north-to-south gradient in the DNASE1 allele.     

Multiple forms of choline acetyltransferase in several species demonstrated by isoelectric focusing

1. The behaviour of choline acetyltransferase from pigeon, guinea-pig, rat and cat brain on isoelectric focusing was studied. 2. Choline acetyltransferase from pigeon and guinea-pig brain showed single peaks with isoelectric points at pH6.6 and 6.8 respectively. Only one molecular form of the enzyme was therefore detected in these species. 3. Three peaks of choline acetyltransferase activities with isoelectric points 7.3-7.6, 7.7-7.9 and 8.3 were obtained with enzyme preparations from rat brain. 4. The separate identities of each of the three forms were confirmed by refocusing. 5. Choline acetyltransferase activity from a high-speed supernatant of rat brain homogenate was distributed similarly to a partially purified enzyme preparation from rat brain in the isoelectric gradient. 6. The enzyme activities from cat brain were separated into two distinct peaks with isoelectric points 7.0 and 8.4, and a possible third peak with isoelectric point 7.6. 7. The two main peaks showed considerable differences in stability on storage, and their identities were confirmed by refocusing. 8. The distribution of the enzyme activities was unaltered by isoelectric focusing in the presence of 3m-urea. 9. The apparent K(m) for choline of choline acetyltransferase from rat, cat and guinea-pig brain was 0.8mm, whereas for the pigeon enzyme it was 0.4mm.     

Deoxynivalenol and Its Monoacetate: New Mycotoxins from Fusarium roseum and Moldy Barley

Five kinds of thermo-labile antigens (TLA a, TLA b, TLA c, TLA d and TLA e) of yeast cell surface were isolated. Each TLA was homogeneous on polyacrylamide gel electrophoresis. The molecular weights of TLA a, TLA b, TLA c, TLA d and TLA e were 68,000, 86,000, 68,000, 170,000 and 82,000, respectively, as estimated by gel filtration on Sephadex G-200. When gel electrophoresis was performed in 0.1% sodium dodecyl sulfate (SDS), TLA a, TLA b, TLA c and TLA e gave a single band of molecular weight at about 62,000, 38,000, 33,000 or 79,000 in each test, whereas TLA d gave two bands of molecular weights at about 81,000 and 40,000. Among the five kinds of antigens, only TLA e contained 9.7% sugar, the other antigens containing no sugar moiety. TLA a, TLA c, TLA d and TLA e did not give a precipitin line against anti-TLA antisera when heated at 70°C for 10 min, whereas TLA b lost antigenicity by heating at 90°C. the isoelectric points for the five kinds of antigens were at pH 6.4 (TLA a), pH 6.1 (TLA b), pH 5.2 (TLA c), pH 4.7 (TLA d) and pH 4.0 (TLA e). All antisera specific to each TLA agglutinated fresh yeast cells, suggesting that these five kinds of antigens are located on the cell surface.     

Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin. II. Electrostatic interactions in individual fatty acid binding sites

13C NMR chemical shift results as a function of pH for a series of carboxyl 13C-enriched saturated fatty acids (8-18 carbons) bound to bovine serum albumin (BSA) are presented. For octanoic acid bound to BSA (6:1, mol/mol), the chemical shift of the only FA carboxyl resonance (designated as peak c), plotted as a function of pH, exhibited a complete sigmoidal titration curve that deviated in shape from a corresponding theoretical Henderson-Hasselbach curve. However, FA carboxyl chemical shift plotted as a function of added HCl yielded a linear titration curve analogous to those obtained for protein-free monomeric fatty acid (FA) in water. The apparent pK of BSA-bound octanoic acid was 4.3 +/- 0.2. However, the intrinsic pK (corrected for electrostatic effects resulting from the net positive charge on BSA) was approximately 4.8, a value identical to that obtained for monomeric octanoic acid in water in the absence of protein. For long-chain FA (greater than or equal to 12 carbons) bound to BSA (6:1, mol/mol), chemical shift titration curves for peak c were similar to those obtained for octanoic acid/BSA. However, the four additional FA carboxyl resonances observed (designated as peaks a, b, b', and d) exhibited no change in chemical shift between pH 8 and 3. For C14.0 X BSA complexes (3:1 and 6:1, mol/mol) peaks b' and a exhibited chemical shift changes between pH 8.8 and 11.5 concomitant with chemical shift changes in the epsilon-carbon (lysine) resonance. In contrast, peaks c and d exhibited no change and peak b only a slight change in chemical shift over the same pH range. We conclude: the carboxyl groups of bound FA represented by peaks a, b, b', and d were involved in ion pair electrostatic interactions with positively charged amino acyl residues on BSA; the carboxyl groups of bound FA represented by peak c were not involved in electrostatic interactions with BSA; the similarity of the titration curves of peak c for BSA-bound octanoic acid and long-chain FA suggested that short-chain and long-chain FA represented by peak c were bound to the same binding site(s) on BSA; bound FA represented by peaks b' and a (but not d or b) were directly adjacent to BSA lysine residues. We present a model which correlates NMR peaks b, b', and d with the putative locations of three individual high-affinity binding sites in a three-dimensional model of BSA.     

Evidence for the phosphorylation of a proenkephalin-derived peptide, peptide B

The potential phosphorylation of a proenkephalin-derived peptide, Peptide B, was investigated in primary cultures of bovine adrenal chromaffin cells and fresh adrenal medullary tissue. Cultures were labeled with [32P]phosphate for 24 h and extracts subjected to immunoprecipitation using affinity-purified anti-serum directed against the carboxyl terminus of Peptide B. A 4.6-kDa-labeled peptide was observed in autoradiograms of immunoprecipitates separated by sodium dodecyl sulfate-polyacrylamide electrophoresis; this peptide was not observed when excess antigen was present during the immunoprecipitation. Radioimmunoassay of extracts prepared from adrenal medullary tissue and separated by isoelectric focusing revealed the presence of four isoelectric forms of Peptide B-immunoreactive peptides; these peptides also exhibited Met-enkephalin-Arg-Phe immunoreactivity. The isoelectric points of these peptides (4.5, 4.3, 4.1, and 3.9) were consistent with the predicted pI values for phosphorylated derivatives of Peptide B. Treatment of samples with alkaline phosphatase prior to isoelectric focusing resulted in the conversion of the more acidic forms to the least acidic form. The presence of phosphate in the more acidic peaks was additionally verified by isoelectric focusing of 32P-labeled immunoprecipitates; the pI values of the radioactive peptides corresponded precisely to the peaks of immunoreactivity. In adrenal medullary tissue, the relative contributions of the various phosphorylated species to the total Peptide B immunoreactivity were as follows: unphosphorylated form, 13%; singly phosphorylated, 31%; doubly phosphorylated, 37%; and triply phosphorylated, 17%. Thus more than 85% of the Peptide B molecules present in the bovine adrenal medulla are phosphorylated.     

Cytochrome c oxidase from bakers' yeast. III. Physical characterization of isolated subunits and chemical evidence for two different classes of polypeptides.

Earlier studies have shown that cytochrome c oxidase from bakers' yeast is an oligomeric enzyme which contains three polypeptides (I to III) synthesized on mitochondrial ribosomes and four polypeptides (IV to VII) synthesized on cytoplasmic ribosomes. These polypeptide subunits have now been isolated by a simple protocol which utilizes differences in polypeptide charge, solubility, and size. Their molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, gel filtration in the presence of guanidine hydrochloride, and amino acid analysis were: I, 40,000; II, 33,000; III, 22,000; IV, 14,500; V, 12,700; VI, 12,700; and VII, 4,600. All seven polypeptide subunits exhibited acidic isoelectric points; cytoplasmically made subunits were more acidic than mitochondrially made ones. The amino acid composition of two mitochondrially made subunits and two cytoplasmically made subunits was determined. The two mitochondrial translation products, I and II, contained only 34.7% and 42.1% polar amino acids, respectively, whereas the two cytoplasmic translation products, IV and VI, contained 48.3% and 49.3%, respectively. This agreed with the observation that Subunits I and II are very insoluble, requiring detergents for solubility, whereas Subunits IV and VI are water-soluble in the absence of any added detergent. These results indicate that the cytochrome c oxidase subunits synthesized on mitochondrial and cytoplasmic ribosomes are fundamentally different in size, isoelectric properties, and hydrophobicity. They also suggest the possibility that at least some of the mitochondrially made subunits are buried in the lipid phase of the mitochondrial inner membrane.     

Electrophoretic Differences in the Capsid Proteins of Simian Virus 40 Plaque Mutants

The structural proteins of three mutants of simian virus 40 (SV40) which differ in plaque size, temperature sensitivity, oncogenicity, host cell restriction, and immunological properties were studied. The polypeptide components of these SV40 strains could not be distinguished by their polyacrylamide gel electrophoretic patterns. When the dissociated virions of two of the mutants were analyzed by the isoelectric focusing technique in a urea gradient, the capsid protein peaks were found to differ significantly in their isoelectric points. The capsid protein of the small-plaque mutant had an isoelectric point of pH 6.51 as compared with pH 6.28 for the large-plaque strain. Isoelectric focusing of the isolated capsid protein revealed three components, a single major subunit and two minor forms. The coat proteins of two of the mutants, small-plaque and minute-plaque strains, were indistinguishable by this technique. The capsid protein peaks obtained by isoelectric focusing were further analyzed by polyacryalmide gel electrophoresis.