Isoelectric Species Research Articles

Actin Amino-Acid Sequences. Comparison of Actins from Calf Thymus, Bovine Brain, and SV40-Transformed Mouse 3T3 Cells with Rabbit Skeletal Muscle Actin

Actin was purified from calf thymus, bovine brain and SV40-transformed mouse 3T3 cells grown in tissue culture. Isoelectric focusing analysis showed the presence of the two actin polypeptides beta and gamma typical for non-muscle actins in all three actins. Tryptic and thermolytic peptides accounting for the complete amino-acid sequence of the cytoplasmic actins were separated and isolated by preparative fingerprint techniques. All peptides were characterized by amino-acid analysis and compared with the corresponding peptides from rabbit skeletal muscle actin. Peptides which differed in amino-acid composition from the corresponding skeletal muscle actin peptides were subjected to sequence analysis in order to localize the amino-acid replacement. The results obtained show that all three mammalian cytoplasmic actins studied contain the same amino-acid exchanges indicating that mammalian cytoplasmic actins are very similar if not identical in amino-acid sequence. The presence of two different isoelectric species beta and gamma in cytoplasmic actins from higher vertebrates is acccounted for by the isolation of two very similar but not identical amino-terminal peptides in all three actin preparations. The nature of the amino-acid replacements in these two peptides not only accounts for the different isoelectric forms but also shows that beta and gamma cytoplasmic actins are the products of two different structural genes expressed in the same cell. The total number of amino-acid replacements so far detected in the comparison of these cytoplasmic actins and skeletal muscle actin is 25 for the beta chain and 24 for the gamma chain. With the exception of the amino-terminal three or four residues, which are responsible for the isoelectric differences, the replacements do not involve charged amino acids. The exchanges are not randomly distributed. No replacements were detected in regions 18--75 and 299--356 while the regions between residues 2--17 and 259--298 show a high number of replacements. In addition documentation for a few minor revisions of the amino acid sequence of rabbit skeletal muscle actin is provided.

The major proteins of the Escherichia coli outer cell-envelope membrane. Heterogeneity of protein I.

One of the major proteins of the Escherichia coli outer cell envelope membrane, protein I, can be separated electrophoretically into protein components Ia and Ib. Strain differences exist regarding presence or absence of component Ib and this component can selectively be lost by mutation to resistance against a phage. Both components Ia and Ib are further heterogeneous isoelectrically, and both together may contain at lease six separable isoelectric species. As judged by analysis of their cyanogen bromide fragments, Ia and Ib are almost identical concerning their primary structure; the difference (charge only or size and charge) was located in a part of the protein that does not correspond to the C-terminal or N-terminal regions. Components Ia and Ib thus represent essentially the same polypeptide and they may arise by a modification process in vitro or the existence of two almost identical genes.

Subunit structure of the 12S protein from seeds of Brassica juncea

The 12S globulin from Brassica juncea seeds has been dissociated by 6 M urea: 0.1 M mercaptoethanol and the resultant products have been partially separated by preparative isoelectric focusing. Species that have the isoelectric points 4.75, 5.15, 5.40, 6.25, 6.70, 7.00, 7.20, 7.70, 8.30, 8.90, and 9.15 were detected. The results of poly aery lamide-gel electrophoretic analysis and the amino acid composition of the fractions suggested that the isoelectric species were distinct subunits.

Incremental combining site filling of anti-polypeptide antibodies

Antibodies involed in the ‘calcium dependent’ reaction of (Glu 60Ala 30Tyr 10) n anti-(Glu 60Ala 30Tyr 10) n have been fractioned by sequential precipitation with the glutamyl containing cross reacting polypeptides (Glu) n , (Glu 90Ala 10) n , (Glu 60Ala 40) n , in that respective order, and finally antigen. Isoelectric focusing and hydrogen exchange experiments have been performed on the parent preparation and the fractionated subpopulations. Although it had been reported earlier that the preparation contained four isoelectric species, as determined in gel experiments, the liquid column studies on the parent preparation and the subpopulations indicate that as many as 25 antibodies which are involved in calcium dependent interactions are elicited by (Glu 60Ala 30Tyr 10) n . The immunochemical basis for this heterogeneity of response is considered. From the hydrogen exchange studies done at high levels of antigen excess, where it has been shown previously that combining site sensitive hydrogen are blocked from exchange upon interaction with antigen, the extent of interaction of the cross reacting polypeptides with the parent preparation and the subpopulations have been determined. For the subpopulation isolated with poly Glu the hydrogen exchange studies show that 55 per cent of the combining site sensitive hydrogens are affected by interaction with poly Glu, 85% with (Glu 90Ala 10) n , with (Glu 60Ala 40) n and 100 per cent with the immunizing antigen. This incremental combining site filling of this subpopulation by the cross reacting polypeptides correlates with the light transparency of the respective specific precitates. Thus, that formed with poly Glu is transparent while those formed with the other polypeptides have increasing opacity, becoming typically white when formed with the immunizing antigen. The exchange studies done on the subpopulation isolated by precipitation with (Glu 90Ala 10) n indicate that the combining sites of these antibodies are mostly directed against determinants containing Glu and Tyr. For the subpopulation which requires the immunizing antigen for precipitation, the exchange studies show that the combining sites of these antibodies are most likely directed against unique sequences of Glu, Ala and Tyr. By comparing quantitative cross precipitin reaction data on the parent preparation with data obtained by hydrogen exchange, it is found that no correlation exists between the amount of antibody a cross reacting polypeptide precipitates and the percentage of ligand sensitive combining site hydrogens with which it interacts.

Kinetics and Mechanism of Decarboxylation of some Pyridinecarboxylic Acids in Aqueous Solution

The first-order rate constants for decarboxylation of picolinic and five substituted picolinic acids in buffered aqueous solution at 150° and ionic strength 1.0 increase as pH increases above 0, go through a maximum at pH near 1, then level off at about half the maximum. For quinolinic acid at 95° the rate maximum occurs at the isoelectric pH. It is therefore concluded that the anion decarboxylates about half as fast as the isoelectric species. Anion and isoelectric species show similar carboxyl-carbon kinetic isotope and substituent effects, so they probably decarboxylate by similar mechanisms. The methyl betaine of picolinic acid decarboxylates about 200 times faster than the anion. From these facts it is concluded that the isoelectric species which decarboxylates is probably zwitterion rather than neutral acid, and that anion and zwitterion decarboxylate by loss of carbon dioxide to form 2-pyridyl carbanion and ylid, respectively. The slower decarboxylation of isoelectric species compared to betaine suggests that only a small fraction of the isoelectric species is zwitterion. However, qualitative estimates from spectra of the fraction of zwitterion present in the isoelectric species at 150° suggest that this cannot be the whole explanation. The relative reactivities of the 2-and 4-positions are similar for decarboxylation and hydrogen exchange in pyridinium ions, but not in the unprotonated species.

Comparison of some sex-dependent proteins in the adult male rat

An hepatically synthesized sex-dependent protein ( α 2u), isolated from male rat urine, has been compared with other similarly designated rat proteins. Using a specific antiserum prepared against α 2u, immunological analysis revealed no cross reaction with a sex-dependent protein prepared from unsonicated rat liver homogenates. An α-globulin separated from urine by molecular sieving and chromatography on CM-cellulose was shown to be immunologically identical with α 2u. This α-globulin exhibited only a single electrophoretic band (acrylamide gel) whereas α 2u contained multiple species of protein having different isoelectric points. The single isoelectric form obtained from CM-cellulose migrated electrophoretically as did the major α 2u (5.2) component. Further studies showed that CM-cellulose could be used to separate the various isoelectric species of α 2u. Antisera to murine, urinary sex-dependent proteins have been shown to cross react with some urinary proteins in the rat. However, an antiserum to α 2u did not cross react with any murine urinary proteins.

Ornithine Transcarbamylase from Streptococcus faecalis and Bovine Liver: II. MULTIPLE BINDING SITES FOR CARBAMYL-P AND l-NORVALINE, CORRELATION WITH STEADY STATE KINETICS

Abstract The binding of carbamyl-P, l-ornithine, and its competitive inhibitor l-norvaline by ornithine transcarbamylase has been measured by gel filtration at 25° under the conditions used for the kinetic experiments. Carbamyl-P forms a reversible binary complex with the enzyme from both liver and Streptococcus faecalis, whereas l-norvaline is bound tightly only in the presence of carbamyl-P. There is the equivalent for each ligand of one site per monomer. Two dissociation constants, differing 10-fold, are required to describe the data for the binding of both carbamyl-P and norvaline by the S. faecalis enzyme, and three, even more widely separated, for the binding of carbamyl-P to the bovine enzyme. l-Ornithine, which is also poorly bound to the free enzyme, is, in contrast to norvaline, tightly bound in the presence of phosphate. The initial rate data for the S. faecalis enzyme in both the forward and reverse reactions at pH values between 6.6 and 8.8 can be fitted to the rate equation for the ordered sequential mechanism suggested by the requirements for binding. The dissociation constant for carbamyl-P calculated from the kinetic data is close to the lower of the two dissociation constants into which the binding data was resolved, and the equilibrium constant calculated from the kinetic parameters agrees with that determined directly. But, in spite of the fit of these data to a single set of parameters, the variation of Ki norvaline with the concentrations of norvaline and either carbamyl-P or phosphate in both the forward and reverse reactions suggests that the activity is the sum of the activities of species with different kinetic parameters. The influence of sites that are either not identical or interact anticooperatively on the kinetics is more apparent with the bovine enzyme, for which the double reciprocal plots of activity against the concentration of carbamyl-P are concave downward at every pH studied between 6.1 and 8.9. Vf for the S. faecalis enzyme increases with increase in pH, reaching a plateau at pH 8.4. At this pH the turnover per dimer (80,000 g) is 3,400 s-1 at 25°, Korn 0.7 mm, Kcp 0.29 mm, and Kicp 12 µm. At pH 7.1, the optimum for the reverse reaction, Vr, is 90 s-1, Kp 0.8 mm, Kcit 17 mm, and Kip 8 mm. Vf and Korn were determined for the bovine enzyme at a saturating concentration of carbamyl-P. The variation of Vf with pH conforms to the titration curve for a group with pK 6.65; at the plateau (pH 8.5), the turnover per site (36,000 g) is 187 s-1 at 25°. Korn varies with pH approximately as the concentration of the isoelectric species of ornithine. Kcp decreases with decrease in pH; at pH 8.5 the value estimated from the linear portion of the reciprocal plot at low concentrations is 0.035 mm and from the linear portion at high concentrations, 0.4 mm.

Diacetyl (Acetoin) reductase from Aerobacter aerogenes. Evidence for multiple forms of the enzyme.

A homogeneous preparation of diacetyl (acetoin) reductase from Aerobacter aerogenes has been shown to consist of several isoelectric species. The native enzyme consists of at least 12 species, all possessing enzymatic activity. No differences in the band patterns were observed when curd extract, purified enzyme, purified enzyme in the presence of NAD, and reactivated enzyme from urea treatment, were subjected to isoelectric focusing in polyacrylamide gels. Electrofocusing of the monomers in urea gels gave at least 5 bands. The isoelectric points for the bands were in the range of 5.8 to 6.8.