Electrophoretic Mobility In Agarose Gels Research Articles

Three high molecular weight protease inhibitors of rat plasma. Reactions with trypsin.

We have compared the reactions of trypsin with human alpha 2-macroglobulin (alpha 2M), and three rat plasma protease inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2M. All four of these proteins appear to contain reactive thiol esters. The electrophoretic mobility in agarose gels of human and rat alpha 2M is increased by 1 mol of trypsin, while the mobility of alpha 1M and alpha 1I3 is decreased. Treatment with methylamine causes similar mobility changes, except in the case of rat alpha 2M. Titration of human and rat macroglobulins by repeated small additions of trypsin and by assay of liberated SH groups or enhanced ligand fluorescence revealed a stoichiometry of about 1 mol of trypsin/mol of inhibitor. In contrast, addition of macroglobulin to a fixed amount of trypsin and detection of residual amidase or protease activity revealed a stoichiometry of about 2 mol of trypsin for 1 mol of human alpha 2M, about 1.4 mol for rat alpha 1M, and about 1 mol for rat alpha 2M. One mol of trypsin reacted with 2 or more mol of alpha 1I3 by the criteria of SH groups liberated or protease inhibition. Methylamine-treated rat alpha 2M binds a significant amount of trypsin releasing about 2 mol of SH. Radioactive beta-trypsin was covalently bound to subunits of the purified plasma inhibitors. The Mr of the labeled products with rat and human alpha 2M had molecular weights which suggested trypsin was bound to intact as well as cleaved subunit chains and also to multiple chains via cross-linking. Rat alpha 1M also produced a product which may be an intact subunit alpha chain plus trypsin. Greater than 80% of the trypsin was bound covalently to these inhibitors at low molar ratios.

Tetranitromethane modification of human high density lipoprotein (HDL3): inactivation of high density lipoprotein binding is not related to cross-linking of phospholipids to apoproteins.

Treatment of human high density lipoprotein (HDL) with tetranitromethane (TNM) inhibits its binding to HDL-specific binding sites of cells and isolated membranes. The mechanism of this inhibition, however, is not known; during treatment of HDL with TNM, in addition to the expected nitration of tyrosine residues, cross-linking of lipids to apoproteins and of apoproteins to one another occurs. In order to determine whether the cross-linking of lipids to apoproteins occurs through the carbon-carbon double bonds in the acyl chains, and to determine whether the cross-linking of phospholipids to apoproteins is a possible mechanism of inhibition of binding, we have prepared a reconstituted HDL3 in which the native phospholipids were replaced with dimyristoyl phosphatidylcholine (DMPC). As a control, a reconstituted HDL3 (C-r-HDL3) was also prepared using the total apoproteins and the total lipid constituents of native HDL3. The reconstituted DMPC-containing HDL3 (DMPC-r-HDL3) was similar to native HDL3 and to C-r-HDL3 in its agarose gel electrophoretic mobility, in its chemical composition, and in its binding to rat liver plasma membranes. When treated with TNM, DMPC-r-HDL3, like the native HDL3 and C-r-HDL3, lost its ability to bind to the HDL binding sites of rat liver plasma membranes, as determined by competitive binding assays with 125I-labeled human HDL3 as the tracer. Nitrated DMPC-r-HDL3 contained only traces of phospholipids covalently linked to apoproteins, whereas 21-26% of the total phospholipids were cross-linked to apoproteins of nitrated C-r-HDL3 and nitrated native HDL3.(ABSTRACT TRUNCATED AT 250 WORDS)

On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities.

A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto-correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted.

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity.

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gka and Gkb, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent Km values for MgATP2- and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.

Purification and characterization of bovine lipoproteins: resolution of high density and low density lipoproteins using heparin-Sepharose chromatography.

The selective and reversible adsorption of bovine low density lipoproteins (LDL) by heparin-Sepharose has been exploited as the critical step in a procedure for the preparative isolation of very low density lipoproteins (VLDL)/chylomicrons, LDL, and high density lipoproteins (HDL) from bovine plasma. Molecular size exclusion chromatography and isopycnic density gradient separation steps are also involved in the method described. The resulting HDL and LDL fractions are free from contamination by one another as judged by electrophoretic mobility in agarose gels. The major lipid and apolipoprotein compositions of the three resolved lipoprotein classes have been determined.

Isolation and partial characterization of staphylococcal decomplementation antigen.

A substance with potent decomplementation activity was isolated from staphylococcal culture supernatants by polyethylene glycol precipitation, DEAE-ion-exchange and Sephacryl chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified substance exhibited all the characteristics of the decomplementation antigen (DA) previously detected in unfractionated culture supernatants. It contained glucosamine and phosphorus and was provisionally identified as extracellular, water-soluble teichoic acid of Staphylococcus aureus. DA was entirely resistant towards the action of proteases, DNase, RNase, or lysostaphin and withstood boiling for 30 min. Its electrophoretic mobility in agarose gels at pH 8.7 was approximately double that of human serum albumin. The molecule eluted in a molecular-weight region of 70,000 to 120,000 on Sephacryl S-300 and sedimented as a symmetrical 3 to 4 S moiety in sucrose density gradients. It migrated near the dye front on 12.5% sodium dodecyl sulfate-polyacrylamide gels and remained undenatured after boiling in sodium dodecyl sulfate. DA formed a symmetrical immunoprecipitate upon crossed immunoelectrophoresis against pooled human immunoglobulin G. It was identified as the major extracellular antigen present in unfractionated S. aureus culture supernatants that is precipitable by naturally occurring human immunoglobulin G antibodies. Immune complexes forming between DA and human immunoglobulin G exhibited an extraordinary capacity to activate the classical complement pathway. Micro- or nanogram amounts of purified antigen added to antibody-containing human serum effected rapid and complete consumption of C3, C4, and C5. The biochemical and biological properties of DA single out this molecule for an important role in suppressing the opsonizing activity of host complement through induction of abortive complement consumption in the fluid phase.

Characterization of transglutaminases in normal and malignant human leukocytes.

Using the fluorescent activity staining procedure, transglutaminases in human monocytes, granulocytes and lymphocytes have been characterized with respect to agarose gel electrophoretic mobility and thrombin dependence. A thrombin dependent transglutaminase was found in concanavalin A stimulated peripheral monocytes. The electrophoretic mobility of this zymogen and of platelet and plasma factor XIII was similar. With stimulated peripheral lymphocytes a similar pattern was observed. However, the potential transglutaminase activity in the lymphocyte factor XIII was lower than that in monocytes. Similar results were obtained when lymphocytes from chronic myeloid and chronic lymphocytic leukaemia patients were examined. Quantitatively, the transglutaminase activity in the leukaemic cells was estimated to be lower than the activity in normal cells. With respect to agarose gel electrophoretic mobility, a similar transglutaminase was found in concanavalin A stimulated human peripheral granulocytes. Although electrophoretically clearly separated from tissues transglutaminase, the granulocyte enzyme did not seem to require thrombin for activation.

Comparative study of the U1, U4, and U7 strains of tobacco mosaic virus

Experiments were undertaken to compare three strains of tobacco mosaic virus (TMV) belonging to the tobacco group 1 of the tobamoviruses. Although the U1, U4, and U7 strains of TMV differ significantly in virulence, it was shown that these strains are very closely related. No difference was detected in the major spots of the coat protein tryptic maps and the virions from the three strains shared the same electrophoretic mobility in agarose gels with different buffer systems. The RNA fingerprints of U1 and U7 were indistinguishable and the largest T1 omega fragments of the three strains were identical in size. However, the avirulent U4 RNA fingerprint exhibited a single spot mobility difference from the U1 and U7 RNA fingerprints. This spot corresponded to the T1 phi fragment. This unique fragment is located in the so-called l2 gene between nucleotides 976 and 992 from the 3′ end. This suggests and extends the proposal that the l2 gene is most probably implicated as a factor in the control of virulence (symptomatology).

Flipping of cloned d(pCpG)n.d(pCpG)n DNA sequences from right- to left-handed helical structure by salt, Co(III), or negative supercoiling.

Negative supercoiling of plasmid DNAs containing 24--42 base pairs of alternating d(pCpG) inserts is shown to cause the flipping of the helical hand of the inserts from right to left under physiological conditions. For a negatively supercoiled DNA with a fixed linking number, this flipping reduces its superhelicity and, therefore, is accompanied by a shift of its electrophoretic mobility in agarose gel. Quantitation of the mobility shifts indicates that essentially the whole stretch of contiguous alternating d(pCpG) flips to the left-handed helical form when the negative superhelical density (specific linking difference) is greatest than or equal to 0.03.

Glutaminyl-peptide gamma-glutamyltransferase dependence of the uptake of trypsin-alpha-macroglobulin complexes by macrophages.

The canine alpha-macroglobulin 125I-trypsin complexes were prepared and exposed to canine alveolar macrophages. The binding of the complexes to cells was time- and dose-dependent. A rapid uptake and degradation of the bound complexes was evidenced by the finding of less than 20% cell-bound radioactivity after a 4 h incubation at 20 degrees C. The canine alveolar macrophages contain a glutaminyl-peptide gamma-glutamyltransferase which shows slightly retarded agarose gel electrophoretic mobility as compared to the respective enzymes from tissues of other species, such as guinea pig and man. Evidence is presented that the binding and degradation of trypsin alpha-macroglobulin complexes by macrophages is dependent on this gamma-glutamyltransferase. Monodansylthiacadaverine, a strong inhibitor of this enzyme, blocks the binding of trypsin-alpha-macroglobulin complexes to macrophages and (probably as a consequence of this) degradation of the complexes. Furthermore, this gamma-glutamyltransferase is a calcium-dependent enzyme and the process of binding trypsin-alpha-macroglobulin complexes to the macrophages was likewise found to be calcium-dependent.

Cleavage map of bacteriophage T4 cytosine-containing DNA by sequence-specific endonucleases SalI and KpnI.

Cytosine-containing T4 DNA from endoII- endoIV- dCTPase- alc2 phage grown in a sup+ rB- mB- host is cleaved by endo R.EcoRI and endo R.HindIII to greater than 40 fragments and by endo R.SalI and endo R.KpnI to 8 and 6 fragments, respectively. The latter two fragment sets have been correlated to each other to produce a cleavage map of the genome. The sum of the molecular weights of the fragments calculated from electrophoretic mobility in agarose gels yields a genome molecular weight for cytosine-containing T4 DNA of 105 x 10(6).

Preparation and characterization of form V DNA, the duplex DNA resulting from association of complementary, circular single-stranded DNA

Complementary circular single strands of plasmid PβG or bacteriophage PM2 DNA but not of single-stranded φX174 DNA associate under hybridisation conditions, giving rise to a two-stranded complex. This DNA, which we call form V, has well-defined physico-chemical properties. It sediments as a sharp peak in neutral sucrose gradients; its electrophoretic mobility in agarose gels is between that of covalently closed (form I) and denatured DNA. In the electron microscope form V appears as highly folded duplex molecules indistinguishable from form I. However, increasing concentrations of ethidium bromide which lead to relaxation and recoiling of form I DNA have no comparable effect upon form V. At 260 nm form V PβG DNA has a hypochromicity of 18.6%, as compared to 23.4% in the case of PβG form II DNA and 10.5% in the case of single-stranded φX174 DNA. The thermal melting of form V is non-cooperative with gradual increase in absorbance similar to that of single-stranded DNA. The circular dichroism spectrum of form V DNA differs from that of form I, circular nicked (form II) and single-stranded φX174 DNA in that it shows a negative band at 295 nm and a shift for the main positive band from 273 to 266 nm. We propose that form V consists of right-handed Watson-Crick type double-helices which are compensated by an equal number of left-handed duplex turns and negative supercoils. Wo cannot decide whether left-handed duplex turns are stabilised by base-stacking and hydrogen bonding, as for example in the models described by Rodley et al. (1976) or Sasisekharan & Pattabiraman (1976), or whether they are merely compensatory turns without inherent stability.

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and Hölzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.

Evidence that a separate particle containing B-apoprotein is present in high-density lipoproteins from perfused rat liver

High-density lipoproteins (HDL) (1.075 < d < 1.175) from perfusates of rat liver, unlike those of blood plasma, contain protein with the properties of B-apolipoprotein. This protein remains near the origin upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, has β electrophoretic mobility in agarose gel, is insoluble in tetramethylurea, and precipitates with antisera to the B-apoprotein isolated from low-density lipoprotein. B-apolipoprotein in HDL from perfusates binds to concanavalin-A Sepharose and can thus be separated from the characteristic HDL, the chemical and physical properties of which are otherwise preserved. These observations suggest that in addition to the discoidal lipoproteins, another particle that contains B-apoprotein exists in HDL of perfusates.

Identification and quantitation of capsular antigen in capsulated and noncapsulated strains of Haemophilus influenzae type b by crossed-immunoelectrophoresis.

Sonicated preparations of capsulated Haemophilus influenzae type b, two of its spontaneous mutants, one containing patches of capsules (class I variant) and the other noncapsulated (class II variant), and a noncapsulated strain of H. influenzae type d were analyzed by crossed-immunoelectrophoresis using unadsorbed antiserum to capsulated H. influenzae type b. Twenty common antigens were present in all four cultures. Two type b-specific antigens were also identified in the three H. influenzae type b cultures when antiserum adsorbed with H. influenzae type d sonicates (AdR) was used. One of these, the type b capsular antigen, cross-reacted with an antigen of Bacillus pumilus. Further adsorption of AdR with the B. pumilus sonicate reduced, but did not eliminate, the antibodies to the type b capsular antigen, although all antibodies to B. pumilus were removed. Sonication sheared the type b capsular antigen, resulting in an increase in its electrophoretic mobility in agarose gel. The capsular antigen from all three H. influenzae type b cultures had the same electrophoretic characteristics. Reproducible quantitation of sheared and unsheared capsular antigen was demonstrated by rocket immunoelectrophoresis. As little as 2.5 ng of polyribophosphate pentose was identified and measured. Capsulated H. influenzae type b contained 78 ng of polyribophosphate pentose per mug of cell protein; class I contained 20 ng and class II contained 4 ng. The small amount of type b capsular antigen present in the class II variant may account for its lack of detection in this organism before now.

Relationship between Lipid Composition of Very Low Density Lipoprotein and Electrophoretic Mobility in Agarose Gel

Eleven patients with type IV hyperlipoproteinaemia were investigated. The lipid composition of three very low density lipoprotein (VLDL) subfractions (Sf 100-400, 60-100, and 20-60) obtained by density gradient ultracentrifugation were compared with their electrophoretic mobility in agarose gel. With decreasing Sf values there was cholesterol and phospholipid enrichment relative to triglyceride. All subjects had lipoprotein with beta mobility (LPbeta) in the Sf fraction of 20-60. LPbeta may represent normally occurring intermediary particles.

The relationship between molecular size and electrphoretic mobility in agarose gels as determined from a single population of growing RNA molecules.

Growing RNA molecules obtained from a defined transcription unit, the 75 S RNA transcription unit in Balbiani ring 2 of Chironomus tentans, have been analysed by electrophoresis in agarose. This set of RNA molecules forms a broad distribution of molecules with sizes ranging from very small up to the final product of giant size corresponding to 75 S RNA. The relative molecular weights along the migration path were calculated from a simple activity relationship derived from the expected distribution properties of a single population of growing RNA molecules. It was concluded that there is a linear relationship between the molecular weight and the logarithm for the mobility.

Serum inhibitors in fibrinolysis.

Four protease inhibitors have been identified in human serum and methods for their isolation are described. After removal of the euglobulin fraction, serum was submitted to ion exchange chromatography and gel filtration, and fractions were tested for inhibition of the lysis of plasminogen-deficient fibrin clots by plasmin, trypsin and elastase. In addition inhibitors of plasminogen activation were sought by studying the effects of separated fractions on the lysis of plasminogen-rich fibrin clots by urokinase. Examination by immunophoresis showed that three of the separated inhibitors were alpha2-macroglobulin, alpha1-antitrypsin and inter-alpha-trypsin inhibitor. The fourth antiprotease was a powerful inhibitor of both urokinase-induced and plasmin-induced clot lysis, and was identified as an inter-alpha-globulin from its electrophoretic mobility in agarose gels.

Properties of leucocytic protease

The proteolytic activity of leucocyte extracts was studied with the aid of Sephadex gel filtration, agarose gel electrophoresis and an immunochemical technique. The activity found does not emanate from plasmin. Plasminogen does not occur in any substantial amount in PMN leucocytes. The activity appears to reside in protein of smaller molecular size than albumin. The pH optimum for the bulk of the proteolytic activity with fibrinogen as substrate is pH 8.5. The stability is good at pH 7.4. The electrophoretic mobility in agarose gel in barbiturate buffer varies strongly with the presence or absence of Ca 2+. Sephadex gel filtration of leucocyte extract resulted in a 15-fold increase of the proteolytic activity per mg protein.

Very low density lipoproteins in intestinal lymph: origin, composition, and role in lipid transport in the fasting state.

The transport of endogenous lipids in the lipoproteins of mesenteric lymph was studied in fasting rats with mesenteric lymph fistulas. The lymph was found to contain, in addition to chylomicrons (S(f) >400), a significant amount of another, more dense, triglyceride-rich fraction, the very low density lipoproteins (VLDL), which showed a peak S(f) of 102. The VLDL differed from chylomicrons not only in flotation, but also in per cent lipid composition and electrophoretic mobility in agarose gel. The VLDL fraction was found to contain 47% of the triglyceride and 54% of the cholesterol of fasting lymph and, in the fasting state, was the major lipoprotein species present. When cholestyramine resin was administered intraduodenally, or bile flow was acutely diverted from the intestine, it was demonstrated that the lipids in lymph VLDL, like those in chylomicrons, were derived from the intestine and bile. These data indicate that the VLDL in intestinal lymph are not derived from the plasma but are of intestinal origin. Because certain properties of lymph VLDL were similar to those reported for plasma VLDL (per cent lipid composition, flotation coefficient, and continuing entry into plasma in the fasting state), additional comparisons between these fractions were made. Although lymph VLDL moved to the alpha(2) region in agarose gel, when they were mixed with VLDL-free serum immediately before electrophoresis they showed the alpha(2) mobility of rat serum VLDL. Furthermore, immunoelectrophoretic comparison of partially delipidated lymph and serum VLDL revealed that these fractions shared in common their major apoprotein, and possibly others as well. The fatty acid composition of lymph and serum triglycerides, as determined by gas-liquid chromatography, revealed that although they were generally similar, differences existed which most likely reflected the presence in serum of triglycerides of hepatic origin. These experiments demonstrate the importance of intestinal VLDL in the transport of endogenous lipids in mesenteric lymph in the fasting state. The similarities between intestinal lymph VLDL and plasma VLDL suggest that the latter may be derived in part from the former.