Urea Starch Gel Electrophoresis Research Articles

Origin and evolution of the sulawesi macaques 2. complete amino acid sequences of seven β chains of three molecular types

Seven β chains were identified as the typical molecular types carried by the seven species of Sulawesi macaques based on isoelectric focusing and urea starch gel electrophoresis. These β chains include the β3 chains ofmaura, tonkeana, nigra, andbrunnescens, β1 chains ofhecki andochreata and β5 chain ofnigra. The results of chromatography on cation-exchange and reversed phase columns and the amino acid compositions of the tryptic peptides suggested substitutions at the 9th and 13th amino acids from the N-terminal. Sequence analyses of these seven β chains from the N-terminal to the 18th amino acid and those of purified tryptic peptides from βT3 to βT15 by Edman degradation revealed the following facts: (1) the amino acid sequences of the β3 chains carried by the four species coincided with each other and as did those of the β1 chains of the two named species; and (2) the 9th and 13th amino acids were Lys and Thr in β3, Asn and Asn in β1, and Asp and Thr in the β5 chain, respectively. These three β chains are related with each other by at least two-base changes. The evolution of the β chains of the Sulawesi macaques was inferred to be as follows. (1) The β3 chain might have been dominant β chain in the past among Sulawesi macaques, since peripheral species separately carried this chain; (2) the β1 and β5 chains might have derived from a “missing link” because of more than two-base substitutions between β3 and β1 and between β3 and β5; (3) eight other macaque species, including the lion-tailed macaque (M. silenus), bear Asn and Thr at these two positions, while the Barbary macaque (M. sylvanus) has Thr and Thr; and (4) thus, if the parsimonious rule is followed, the type with Asn-Thr is the most plausible “missing link,” since only the Asn-Thr type can combine these five β chains by minimum one-base change. Two genetic events are postulated in the evolutionary process of the Sulawesi β chains: the first Lys-Thr type (β3) was distributed over the whole island, and next Asn-Thr, the common type in other macaques, produced Asn-Asn (β1) and Asp-Thr (β5).

Haemoglobin and globin synthesis in the isolated primitive and definitive erythroid cells of chicken embryos. Evidence for a non-clonal mechanism at the haemoglobin switch

Primitive and definitive erythroid cells of chicken embryos aged 4-8 days, were separated by unit gravity sedimentation and pulse labelled with [3H]- and [14C]leucine. The haemoglobin and globin synthesis in the cell populations was analysed by chromatofocussing, isoelectric focussing, urea starch gel electrophoresis, and immunofluorescence or radioimmunoassay, using globin specific antibodies. We found that both embryonic and adult alpha globins are present in primitive erythroid cells, but relatively more of the adult alpha-type globins are synthesized in the late primitive erythroid cells. In young definitive erythroid cells exclusively adult alpha-type globins are synthesized. From these findings we conclude that a command to synthesize adult alpha globin is perceived in both cell types at the time of the switch. This supports an environmental model rather than a clonal model of haemoglobin switching.

Nature of the heterogeneity within genetic variants of bovine serum transferrin

A comparison is made of the four main components of an homozygous variant (A or D2D2) of bovine serum transferrin. These are designated I-IV in order of increasing mobility in electrophoresis at pH 7.5. Components I, II, III and IV have 2,2,3 and 3 residues of sialic acid per transferrin molecule and appear to correspond to components 2a, 2b, 3a and 3b respectively of Stratil & Spooner (1971). The difference between components I and II and between III and IV does not reside in sialic acid differences. On the basis of peptide maps of reduced carboxamidomethylated components, urea-starch gel electrophoresis and quantitative sequence studies, it is concluded that components II and IV have a scission in the peptide chain. By homology with the sequency of MacGillivray et al. (1977) for human serum transferrin it is suggested that the scission occurs between residues 55 and 54 from the C-terminus and this portion of the chain has a 'molecular' weight of ca. 6000. The implications are briefly discussed.

Difference between the Hb C variants in Brahman and in indigenous Southern African cattle breeds

SummaryHaemoglobin polymorphism in Brahman cattle and seven Southern African cattle breeds was investigated by means of starch gel electrophoresis. A difference was found between the migration rates of the Hb C of Brahman cattle and that of the indigenous Southern African cattle breeds, showing that these are actually two separate variants. We suggest that the faster migrating type, which occurs in Brahman cattle, should be called Hb C, while the slower migrating type of the Southern African breeds, should be called Hb I.A comparison of the migration of the α and β chains of all the variants we encountered, including foetal haemoglobin, was carried out by means of starch gel electrophoresis in urea at acid and alkaline pH levels. Evidence was found of a difference in mobility of the non‐a chains of haemoglobin A, B, C, I and F at different pH levels while no difference was detected in the migration rate of their respective α chains. These results confirm the theory that genetic variation is restricted to the non‐α chain of bovine haemoglobin.Progeny studies on 225 families confirm that the Hb I variant is allelic to Hb A and Hb B variants. The gene frequencies have been calculated on the basis that Hb I and Hb C are allelic at the β locus. The distribution of the different phenotypes is in accordance with this theory, assuming that the populations obey Hardy‐Weinberg equilibrium.

Structure of bovine casein oligomers

We have characterized the size, molecular weight, and composition of the oligomeric particles produced by dialysis of bovine casein micelles against solutions lacking calcium ion. The particles were stabilized against further dissociation after dialysis by glutaraldehyde fixation. The progress of the dissociation was monitored by Biogel A-15 gel permeation chromatography, sucrose density gradient ultracentrifugation, inelastic laser light scattering, sedimentation velocity and equilibrium, and urea starch gel electrophoresis. The casein oligomers isolated after dialysis against either 0.5 m NaCl/SMUF A/azide or calcium-free SMUF/ azide have a hydrodynamic radius of about 5.5 nm and a molecular weight of about 95,000 corresponding to roughly four casein monomers (SMUF, simulated milk ultrafiltrate). The oligomers are highly hydrated and contain one-third of the calcium ion found in native micelles. During the course of dialysis, the micelles gradually break down into a broad distribution of intermediatesized particles and then into the oligomers described above.

The in <i>vitro</i> Effect of Analgesics on the Electrophoretic Pattern of the Red Cell Membrane Proteins

The <i>in vitro </i>effect of acetylsalicylic acid, phenylbutazone and indomethacin on the electrophoretic pattern of the red cell membrane proteins is described by using the vertical urea starch-gel electrophoresis. Normally 8 bands are visible. Acetylsalicylic acid and phenylbutazone bring about the disappearance of 5 bands and indomethacin that of 6 bands.

Studies on Fd-fragments of human immunoglobulins: II. The preparation and characterization of peptic (Fab′) 2-fragments and an Fd′-fragment of IgA-myeloma proteins

Pepsin digestion of unfolded human 7 S IgA1 myeloma proteins and of an unfolded IgA2 (1+) protein produced, under certain conditions, fragments which are similar with respect to gel filtration. The fragments contained light chains and part of the α-chains as judged by immunodiffusion experiments and urea starch gel electrophoresis. From approximate molecular weight determinations by thin layer gel filtration together with characterization of the fragments by ultracentrifuge experiments, it was concluded that (Fab′) 2-like fragments had been prepared. The nature of the fragments is discussed in relation to the observed antigenic heterogeneity of (Fab′) 2-like fragments from distinct myeloma proteins. Finally an Fd′ α-like fragment could be isolated from one of the studied myeloma proteins; the relevance of this result is discussed in the light of the structure of Fab from human immunoglobulins.

Studies on Human β-d-N-Acetylhexosaminidases: II. KINETIC AND STRUCTURAL PROPERTIES

Abstract β-d-N-Acetylhexosaminidase A and B purified to homogeneity from human placenta have pH optima of 4.4 using 4-methylumbelliferyl-β-d-N-acetylgalactosaminide as substrate. The Km of both hexosaminidase A and hexosaminidase B for this synthetic substrate was found to be 0.5 mm and heat of activation for both the enzymes was 10,500 cal. No effect of any divalent ions studied was observed except for a 25% decrease in the enzyme activity of both hexosaminidase A and B in the presence of 5 mm cupric ion. p-Hydroxymercuribenzoate, 1 mm, almost completely inhibited both hexosaminidase A and hexosaminidase B activity. N-Ethylmaleimide, arsenate, and iodoacetate had no effect on the hexosaminidases. No inhibition of hexosaminidase A or hexosaminidase B was observed in the presence of 30 mm concentration of various anions except for acetate which brought about 75% inhibition of both hexosaminidase A and hexosaminidase B activity. Using Sephadex G-200 filtration, the molecular weight of hexosaminidase A and B was estimated at 140,000. However, using the sedimentation equilibrium technique the molecular weight was found to be 100,000 ± 3,000 and 101,400 ± 4,700 for hexosaminidase A and B, respectively. After S-carboxymethylation, the subunit molecular weight of hexosaminidase A and B was determined using various concentrations of polyacrylamide in disc electrophoresis in the presence of sodium dodecyl sulfate. Hexosaminidase B dissociated into one major subunit corresponding to a molecular weight of 17,000 to 18,000, whereas hexosaminidase A dissociated into one major subunit corresponding to a molecular weight of 17,000 to 18,000 and two more protein bands corresponding to about 35,000 and to 55,000. Similar results were obtained when hexosaminidase A and B were dissociated into subunits by treatment with guanidine hydrochloride or by treatment with maleic anhydride in the presence of sodium dodecyl sulfate and the subunits separated by polyacrylamide gel electrophoresis. In urea-starch gel electrophoresis hexosaminidase A dissociated into three major bands and hexosaminidase B into two bands. In urea-starch gel the electrophoretic mobility of one of the bands of dissociated hexosaminidase A and hexosaminidase B seemed to be very similar or identical. Both hexosaminidase A and hexosaminidase B have blocked NH2-terminal groups. The COOH-terminal amino acids in hexosaminidase A was found to be serine and in hexosaminidase B was found to be aspartic acid or asparagine. The amino acid composition of hexosaminidase A and hexosaminidase B was determined and found to differ.

The molecular basis of the heterogeneity of the MM isozyme of rabbit muscle creatine phosphokinase

Purified rabbit muscle creatine phosphokinase MM isozyme was resolved into a pattern of six subbands by starch gel electrophoresis at pH 6.3 in a Tris-citrate buffer system. Following urea dissociation and denaturation, urea-starch gel electrophoresis resolved six distinct, enzymatically inactive subunits of molecular weight 43 000 daltons as determined by sodium dodecyl sulfate-acrylamide gel electrophoresis. This indicates that six different subunit types are involved in rabbit MM isozyme subbanding. The six subbanded pattern obtained following renaturation and reassociation from urea was identical to that exhibited by the purified, untreated MM isozyme. The exact nature of the subbanding is not clear; but it does not appear to be due to sulfhydryl group interactions or to alternative conformations of a single M-type subunit. Rather, it appears to be due to conformational differences resulting from alterations in primary structure, deamidation or ligand heterogeneity.

Structural and immunological studies of two IgGl-λ myeloma proteins from a single patient

Serum from a patient with multiple myeloma contained two monoclonal IgGl-λ M-components of different electrophoretic mobility. Melphalan therapy reduced the serum concentration of the slow M-component (average pI 7·1) but had no effect on the fast M-component (average pI 6·5). Conversion of the slow M-component into the fast M-component could not be accomplished by in vitro incubation of the patient's serum. Both components had identical sedimentation coefficients of 6·5 S ruling out the possibility of aggregation of one component to another serum protein. Neither M-component showed antigen-combining specifity. Both isolated chains belonged to the λl subgroup with Gm a −b −f + allotypes and were indistinguishable in their acidic and alkaline urea starch gel electrophoretic patterns. The isolated light chains of both M-components were V λIII[St(−) II, Oz(−)] and were indistinguishable in the first five NH 2-terminal residues ([]-Tyr-Val-Leu-Thr), circular dichroism spectra (240–310 nm), amino acid composition and mobility in acidic urea starch gel[ electrophoresis. However, the light chains had different moobility in alkaline urea starch gel electrophoresis. Since both light chains lacked sialic acid, the differences in electrophoretic mobility can be primarily attricuted to the fact the light chain of the fast M-component had 5–7 more dicarboxylic acid residues than the light chain of the slow M-component, and this difference in primary sequence was also reflected un the peptide maps. The liquid isoelectric focusing elution profile of both M-components indicated that each component resolved into four subfractions in decreasing concentration from cathode to anode and any two successive subfractions differed by 0·1 pH unit. This microheterogeneity may also be due to a variable dicarboxylic acid content and could have arisen by post-synthetic deadmidation. The two M-components differed in their average pI values by 0·6 pH units suggesting at least six charge differences between the two M-components. The presence of the two M-components differing in primary sequence of the light chains could have resulted from either a divergent clone or two clones.

Properties of placental alkaline phosphatase. III. Thermostability and urea inhibition of isolated components of the three common phenotypes

Different heat inactivation rates were found among the three common homozygous human placental alkaline phosphatase phenotypes with respect to component I (the rapidly migrating component). Phenotype I1 was less stable than F1 and S1, while types F1 and S1 exhibited very similar thermostabilities, F1 being slightly more stable than S1. Components I, IIα (the fastest of the slow-moving components), and IIβ (the group of the very slowly migrating components) had different heat stabilities, IIβ being the most stable and I the least stable. Magnesium greatly increased the heat stability of all tested phenotypes. Placental alkaline phosphatase was found to be less heat resistant than reported previously. All phenotypes were equally inhibited over urea concentrations ranging from 0.5 to 8 m. No difference in the inhibition rate was found among components I, IIα, IIβ, and the crude placental butanol extract. The altered electrophoretic pattern of the crude placental extract obtained by urea starch gel electrophoresis was considered as being most likely due to reversible changes in the folding of the molecules.

Physicochemical, Immunological and Structural Studies on Two M-Components from a Single Individual

Abstract Serum from a patient with multiple myeloma contained two elevated monoclonal IgG M-components which differed with respect to electrophoretic mobility. Prolonged melphalan therapy reduced the serum concentration of the slow component (γ-electrophoretic mobility, pI 7.1) to normal levels but had no effect on the fast component (β-electrophoretic mobility, pI 6.8). The two M-components were isolated by column chromatography, on DEAE-cellulose and Sephadex G-200, and by isoelectric focusing. Both isolated heavy chains belonged to γG1 subgroup (a-b-f+ allotypes), possessed glutamic acid as the N-terminal amino acid, and were indistinguishable by acid and alkali urea starch gel electrophoresis, amino acid analysis, peptide mapping, and amide content. The isolated light chains of both M-components were λ(III) [Oz (-), St (-) II], possessed tyrosine as the N-terminal amino acid and were indistinguishable by their initial N-terminal sequence [tyr, valleu, leu, thr], circular dichroism spectra (240 to 310 nm), amino acid analysis, and mobility in acid urea starch gel electrophoresis.

Microheterogeneity of Light Chains Derived from a Biclonal Gammopathy

Abstract A patient, Mi., with a biclonal gammopathy of the λ IgM-λ IgG type was studied. Antisera made against the λ light chains showed idiotypic specificity that was common to both light chains so that by this criterion the light chains derived from the IgG and IgM proteins were identical. However, acid urea starch gel electrophoresis indicated a slight but definite difference in mobility, the light chain derived from the IgG having a slightly more cathodic migration. Amino acid composition analysis of the light chains revealed differences between the two light chains in that the IgM λ chain had one mol less Arg and 0.7 mol less Glu and 1 mol more alanine and tyrosine. All these differences were significant at the 0.05 percentile level. Peptide mapping revealed a single peptide difference in these two preparations. The IgG λ chain had a single spot, which was missing from the IgM and on amino acid analysis was shown to be free Arg.

Comparative studies of lens α-crystallin from eight species

1.1. The cross-reactions of α-crystallins from bovine, sheep, rabbit, human, monkey, rat, chicken and dogfish lens were studied by the quantitative complement fixation test using rabbit antisera specific for bovine α-crystallin.2.2. By this method, the bovine, sheep, and rabbit α-crystallins appeared to be identical, while the human, monkey and rat showed a somewhat lower degree of complement fixation with the antibovine α-crystallin sera. The chicken α-crystallin showed approximately 40 per cent cross-reactivity with the bovine protein, and there was no detectable cross-reaction in the case of dogfish α-crystallin.3.3. Excluding dogfish, the electrophoretic patterns of different α-crystallins in 8 M urea starch gel electrophoresis at pH 3·0 were similar.

Differences Between Calf and Adult Bovine Rennet

Activated enzyme extracts of adult bovine abomasum (adult bovine rennet) tissues contained 5 to 15% milk clotting activity attributable to pepsin. Starch gel electrophoresis in urea and mercaptoethanol revealed different patterns than for calf rennet extract. Differences also were in casein-agar gel diffusion patterns at pH 6.8. Lipolytic activities were similar. Proteolytic activity on casein substrate at pH 9.0 was evident in adult bovine but not in calf rennet or in swine pepsin. However, milk clotting activities in adult bovine and calf rennets were destroyed at comparable rates at pH 9.0. Proteolytic activity of adult bovine rennet was significantly reduced by milk clotting activation at pH 2.0. Casein-agar gel diffusion at pH 9.0 was useful in detecting blends of adult bovine and calf rennet. Adult bovine rennet could be detected at 1% concentration. Accelerated ripening and proteolytic activity studies of adult bovine and calf rennet at pH 5.2 indicated possible differences in their effects upon the aging properties of cheese.

Chemical differences of adult, fetal and hypogammaglobulinemic IgG immunoglobulins

Heavy and light chains of IgG molecules from pooled normal adult sera, pooled normal fetal sera, and pooled hypogammaglobulinemic sera were studied by means for starch gel electrophoresis in hypertonic urea. Differences were demonstrated among the heavy chain preparations. These data indicated that: 1. (1) The transfer of IgG molecules across the placenta has a selective nature. Not all species of material IgG pass equally well into the fetal circulation. IgG2 subclass immunoglobulins were detected in the fetal serum in only trace amounts. 2. (2) Neonatal IgG molecules are not entirely composed of molecules passively transferred from the mother. 3. (3) IgG molecules from hypogammaglobulinemic patients are less heterogeneous than those from normal people.

Application of a modified technique to study urinary immunoglobulin light chain and whole IgG excretion in normal human subjects and patients with immune-related disorders.

Abstract While urinary L-chain and IgG excretion has been well studied in normal human subjects, it has received little attention in disease, in large part because of lack of a feasible technique for studying large numbers of patients. The present report describes a simplified technique performed on 350 ml. of urine and involving ultrafiltration, radial immunodiffusion, batching with DEAE Sephadex, and acid 8M urea starch gel electrophoresis. Application of the technique to 10 normal and 69 abnormal subjects free of gross clinical proteinuria revealed elevated L-chain excretion in none of 5 patients with chronic lymphatic leukemia (CLL), 4 of 7 with lymphosarcoma (LSC), 5 of 5 with acute infectious mononucleosis, 2 of 5 with metastatic adenocarcinoma, none of 8 with idiopathic acquired autoimmune hemolytic anemia, 1 of 3 with idiopathic thrombocytopenic purpura, 1 of 5 normally pregnant women, 1 of 6 previously Rh-isosensitized pregnant women, and 9 other patients with a variety of disorders. Urinary L-chain paraproteins and abnormal k:λ ratios were present in 2 patients with CLL and 3 with LSC. Elevated urinary IgG excretion frequently accompanied elevated L-chain excretion. Albumin excretion was essentially normal in all patients. It is concluded that despite absence of clinical proteinuria, abnormal urinary L-chain and IgG excretion are frequently present in malignant lymphoid disorders, regularly present in infectious mononucleosis, and can be present in a variety of other immune-related diseases. Further work is needed to define more clearly the precise origin of urinary L-chain and the mechanisms governing its control before its full clinical value will be known.

Immunochemical Relationship between Soluble and Insoluble Lens Proteins

The relationship between soluble and insoluble proteins in the bovine, human and dogfish lens has been studied by immuno-electrophoresis and urea starch gel electrophoresis at pH 3.0. The result from the immuno-electrophoresis experiment indicated that insoluble protein derived from bovine and human lens is composed of <i>α</i>- and <i>β</i>-crystallins while that obtained from dogfish lens contains <i>γ</i>- and <i>β</i>-crystallins. The urea starch gel electrophoretic patterns of <i>α</i>-crystallin and insouble protein in bovine and human lens showed considerable similarity, however, those of dogfish <i>γ</i>-crystallin and dogfish insoluble protein showed significant differences in their mobilities. The cross-reaction between bovine <i>α</i>-crystallin and bovine insoluble suspension was studied by the quantitative complement fixation test, using rabbit antisera to bovine <i>α</i>-crystallin. The results indicated that 5 to 10% of the total insoluble protein has <i>α</i>-crystallin in a form available for the reaction with the antibody. Similar results were observed when dogfish <i>γ</i>-crystalin and dogfish insoluble suspension was studied.

The Subunit Structure of Chick Lens Beta-Crystallins

The subunit patterns of chick lens β-crystallin was analyzed by urea-starch gel electrophoresis at pH 3.1 and 8.6. Similarities and differences were noted for the different β-crystallins in this regard, and this information may explain the electrophoretic heterogeneity and immunologic cross-reactivity of lens β-crystallins. It was also shown that the subunit pattern of lens β-crystallin is dependent on pH and ontogeny.

Properties of halves of immunoglobulin light chains.

Light polypeptide chains from both normal human gammaG immunoglobulin and Bence-Jones proteins can be cleaved into halves by limited proteolysis with trypsin, pepsin, or papain. The fragments were obtained in yields of up to 22 per cent, had molecular weights of 10,000 to 11,000, and were shown by amino acid analysis and antigenic analysis to correspond to variable or constant regions of light chains. Starch gel electrophoresis in urea suggested that each half consisted of a single polypeptide chain. A fragment from the urine of a myeloma patient corresponded almost exactly to the constant half of a gamma-chain and had a compact shape (Stokes radius of 16 A; frictional ratio of 1.1). Similar Stokes radii were estimated both on fragments from normal urine and fragments produced by proteolysis of normal light chains. The results are consistent with the view that most urinary fragments have a catabolic origin and suggest that there is a small stretch of polypeptide chain between the compact V and C regions of light chains which is particularly susceptible to proteolysis. This lends support to the hypothesis that immunoglobulin chains consist of globular domains connected by more extended stretches of polypeptide chain.