Sequential Gel Research Articles

Urinary excretion patterns of sialoglycoconjugates in patients with mucolipidosis

Excessive urinary excretion of sialoglycoconjugates was observed in 6 patients with mucolipidosis. Urinary sialoglycoconjugates were divided into 9 fractions by sequential gel filtration on Sephadex G-25 and G-50, and by Dowex 1 ion-exchange chromatography. In a comparative study on these fractions it was shown that the fractions rich in sialyl-oligosaccharide contents contributed to the increased urinary excretion of sialoglycoconjugates. The sialyl-oligosaccharides contained in these fractions possessed the core structure of the carbohydrate side chains of glycoproteins with an N-glycosidic linkage, and the sialyl residues in these oligosaccharides were linked to galactosyl residues by an α2,3- or α2,6-linkage. In contrast to the latter type oligosaccharides, the excretion levels of the former varied from patient to patient. The patients with ML II excreted the former oligosaccharide comparable to the excretion levels of the latter. In the patient with ML I the excretion levels of α2,3-linked oligosaccharide was far lower than those of α2,6-linked one, and the patients with macular cherry-red spots and myoclonus syndrome excreted only a trace amount of α2,3-linked oligosaccharide. Mechanisms of these different excretion patterns are discussed.

Isolation and partial characterization of a genus common antigen and species specific antigen of Capnocytophaga.

Sixteen strains of Capnocytophaga were isolated from the pocket of a localized juvenile periodontitis patient. These strains were divided into four groups on the basis of morphological and physiological traits. Strains from group I and group III were identified as C. ochracea and group II as C. sputigena. An antigen common to genus Capnocytophaga was purified utilizing immunoabsorbent chromatography from lysates obtained by sodium dodecyl sulfate treatment of C. ochracea strain S1. An antigen specific to C. ochracea was prepared by sequential gel filtration and preparative isoelectric focusing. The genus common and species specific antigens isolated were immunologically unique and pure when tested by immunoelectrophoresis and immunodiffusion against rabbit antisera prepared to Capnocytophaga and other gram-negative rods. The genus common antigen was susceptible to trypsin and pronase digestion, was soluble in chloroform-methanol, but was unaltered by ribonuclease and deoxyribonuclease treatments and periodate oxidation. Antigenicity of the species specific antigen was destroyed by periodate oxidation. The genus common antigen appeared to be lipid-associated protein, while the species specific antigen consisted mainly of carbohydrate. These specific immunological reagents would be valuable in diagnosing and monitoring diseases.

Purification of a high-affinity discoidin I-binding proteoglycan from axenic Dictyostelium discoideum growth medium

The axenic Dictyostelium discoideum growth medium HL-5, prepared using Difco proteose peptone No. 2, contains an extremely potent inhibitor of the binding of 125I-labeled discoidin I to glutaraldehyde-fixed, cohesive D. discoideum cells. Axenic strain A3 D. discoideum cells bind or internalize the inhibitor during growth in HL-5 medium and subsequently shed or excrete it while differentiating in suspension. The inhibitor has been purified from Difco proteose peptone No. 2 by sequential gel filtration on Sepharose 4B and affinity adsorption using discoidin I-Sepharose. The inhibitor is heterogeneous in molecular weight (4 · 10 5−2 · 10 6), but is relatively homogeneous in density on CsCl density gradients. The size and activity of the inhibitor are resistant to periodate, reduction and maleylation, proteases, nucleases and heating in the absence or presence of sodium dodecyl sulfate. Mild alkali causes a partial reduction in activity and converts the higher molecular weight fraction of the inhibitor to a lower molecular weight. The purified inhibitor contains neutral hexose, hexosamine and amino acid in an approximate molar ratio of 4 : 3 : 2. These and other properties suggest that the inhibitor is an unusual proteoglycan. Certain well-characterized glycosaminoglycans are relatively potent inhibitors of discoidin I binding. The proteoglycan reported here is the most potent discoidin I-binding inhibitor ever identified.

E-Rosette Inhibitory Factor in Sera from Patients with Mycosis Fungoides

Peripheral blood lymphocytes from some of patients with mycosis fungoides disease showed decreased ability to form rosettes with sheep erythrocytes. This decreased percentage of E-rosette forming cells could be normalized when those cells were incubated in culture for 20 hr. Since these data led us to considering a possible inhibitory factor present in patients' sera, we tested their ability to inhibit E-rosetting by T lymphocytes from normal donors, and found that sera from mycosis fungoides patients with low levels of E-rosetting blood lymphocytes showed greater inhibitory effect on E-rosette formation by normal T cells when compared to those either from normal donors or from mycosis patients who had almost normal levels of E-rosetting blood lymphocyte number. The E-rosette inhibitory factor was sensitive to 2-mercaptoethanol treatment and was copurified with serum IgM by ammonium sulfate precipitation and by sequential gel filtrations, suggesting that it might be an anti-T lymphocyte antibody naturally occurring during the disease process.

Soluble C5b-9 complex of guinea pig complement: Demonstration of its heterogeneity and the mechanism of its C9 hemolytic activity as transfer of reversibly bound C9 molecules from the complex

Doubly radiolabeled soluble complex of the late acting components (SC5b-9) was formed by activating guinea pig complement (C)† serum mixed with C5 and C9 each labeled with either 125I or 131I with zymosan. It was isolated by sequential gel filtrations and sucrose density gradient centrifugation. Fractions of the complex obtained by centrifugation were heterogeneous in size, composition and hemolytic activity as C9. The heavier fractions contained four to six molecules of C9 per C5b molecule. The lighter fractions contained two to three C9 molecules to one C5b molecule and had stronger C9 hemolytic activity. On recentrifugation on the same sucrose density gradient, the factions moved to the same positions as on the first centrifugation. Guinea pig SC5b-9 complex bound to the surface of EA, EAC-3, EAC-5, EAC-6, EAC-7 and EAC-8 in similar amounts. Therefore, combination of the complex with these intermediate cells seems to be nonspecific. EAC-7 cells bound to the complex could be lysed by addition of C8. Lighter, hemolytically more active complex purified by recentrifugation was stable when incubated alone in buffer. But when it was incubated with free C9, it released radiolabeled C9, the amount released increasing with increase in the amount of free C9 added to a maximum of 10 to 15% of the C9 in the complex. A minute part of the complex was converted to heavier complex by incorporation of additional C9, but most of the complex did not change in size. Therefore, it did not seem to be an uncompleted complex to be saturated with more C9. The C9 molecules released from the complex were in an active form and could be reincorporatd into SC5b-9 when they were treated with zymosan in fresh guinea pig serum. It is concluded that the C9 hemolytic activity of guinea pig SC5b-9 is exerted by transfer of reversibly bound C9 molecules in the complex to C5b-8 sites on EAC-8 cells, not by the action of the complex in toto.

Abnormal urinary excretion in sialoglycoconjugates in patients with mucopolysaccharidosis.

Abnormal urinary excretion of sialoglycoconjugates was observed in four patients with mucopolysaccharidosis. Urinary sialoglycoconjugates were fractionated into 8 fractions by sequential gel filtration on Sephadex G-25, G-50 and by Dowex 1 ion-exchange chromatography. The comparison of the amounts of these fractions indicated that the fraction rich in mannose and glucosamine contents contributed to the increased urinary excretion of sialoglycoconjugates in patients with mucopolysaccharidosis. The major component of this fraction was disialyl-oroso-N-octaose which was a representative oligosaccharide side chain of glycoproteins with an Asn-GlcNAc linkage. Undoubtedly this abnormality is the secondary lesion of mucopolysaccharidosis, but it is conceivable that the disturbed metabolism of sialoglycoprotein is closely related to the pathogenesis of these diseases.

The sensitivity of gel electrophoresis as a detector of genetic variation.

Three experiments based on an idea of YOUDERIAN have been performed to determine the proportions and kinds of amino acid substitutions that are detected by gel electrophoresis when applied to surveys of protein variation in populations. The experiments involved applying the sequential method of electrophoresis under several conditions of pH and gel concentration to a large sample of human hemoglobins with known amino acid substitutions. In the first experiment, a random sample of 20 different hemoglobin variants was studied, and these were separated into 17 distinct electrophoretic classes by three sequential gel conditions, thus giving a detectability of 85%. A single pass under standard conditions detected eight classes. The second experiment compared groups of substitutions that were chemically identical, but in different positions in the alpha and beta chains, while the third experiment compared pairs of substitutions that were charge equivalent, but chemically different at the same chain position. The sequential method distinguished 90% of all chemically identical substitutions when they were at different chain locations, and four out of five charge equivalent but chemically different substitutions at the same site. Examination of the location of each substitution in the three-dimensional structure of hemoglobins showed that interior substitutions usually are less different from Hb A than are surface substitutions and that local interactions with chain and spatial neighbors are sufficient to distinguish substitutions in very similar positions on the outside of the molecule. The "charge ladder" model of electrophoretic classes is clearly incorrect, and it appears that sequential gel electrophoresis as practiced in our Drosophila surveys has detected a substantial fraction of amino acid substitutions if hemoglobin is regarded as a model. This estimate may be modified as other molecules beside hemoglobin are subjected to similar calibration experiments.

Partial Characterization of Glycopeptides from Culture Filtrates of Fulvia fulva (Cooke) Ciferri (syn. Cladosporium fulvum), the Tomato Leaf Mould Pathogen

Culture filtrates of races 0, 4 and 1.2.3. of Fulvia fulva were fractionated by sequential gel chromatography on Bio-Gel P-2, ion-exchange chromatography on DEAE-cellulose and affinity chromatography on concanavalin A-Sepharose 4B. The fractions were characterized for their carbohydrate, phosphate and protein contents and sugar compositions. d-Galactose, d-glucose and d-mannose were the major sugars with much smaller amounts of d-glucuronic acid, d-galactosamine and d-glucosamine. Phosphate was present as a diester. The carbohydrate was present in a family of glycopeptides of disperse molecular weight, charge and sugar composition. The molecular organization of these polymers was further investigated by mild acid and alkali degradation and by acetolysis of the polysaccharide remaining after these treatments. A possible structure for the polymers is suggested.

Influence of the peptide-chain length on disulphide-bond formation in neurohypophysial hormones and analogues.

(8-Arginine)vasopressin, (8-arginine)vasotocin, oxytocin and oxypressin, the 'ring' derivatives pressinamide and tocinamide, and the extended-chain analogues Pro-Arg-Val-(8-arginine)vasopressin and (8-arginine)vasopressinoyl-Ala-Met-Ala-NH(2), were synthesized by the solid-phase method and purified by sequential gel filtration on Sephadex G-15 in 50% acetic acid and 0.2M-acetic acid. Controlled oxidation of the thiol groups of the reduced peptides obtained after deprotection with sodium in liquid ammonia gave rise to products that depended on the length of the peptide chain: (i) nonapeptides gave monomer and dimer species, (ii) hexapeptides produced mixtures containing higher polymers, and (iii) dodecapeptides gave predominantly monomer with some dimerized material. The evidence suggests that the presence of the acyclic tail tripeptide in the nonapeptide hormones induces a conformation in the preceding hexapeptide that favours the formation of an intramolecular disulphide bond. For (8-arginine)vasopressin, intramolecular disulphide-bond formation is enhanced by extension of the peptide chain from either the N- or the C-terminus. The possible significance of these studies to neurohypophysial hormone-prohormone relationships is discussed.

Starch phosphorylase enzymes in developing and germinating pea seeds

Phosphorylase has been fractionated during development and germination of seeds of smooth and wrinkled-seeded peas. The total phosphorylase levels have been compared. In addition, a number of other pea tissues and other legumes have been examined. Some kinetic properties of the two enzymes present have been measured. Both enzymes have been further purified by affinity chromatography on Sepharose 4B-starch columns and by sequential gel filtration in the absence and presence of amylopectin. The MW and sub-unit structures of the two enzymes have been examined and their possible roles discussed.

Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.

The Molecular Basis of Self-Association of IgG-Rheumatoid Factors

The intermediate complexes, sedimenting between 19S and 6.6S components of normal serum on analytical ultracentrifugation, were purified from plasma of three patients with rheumatoid arthritis. Sequential gel filtration and removal of contaminants by agarose-antibody immunoadsorbents were employed for purification of these complexes. The isolated complexes from the three patients consisted of IgG with k and lambda light chains. Sedimentation equilibrium ultracentrifugation experiments showed that the isolated complexes underwent concentration-dependent self-association, whereby the smallest detectable molecular species had a molecular weight of 292,000. These IgG dimers were formed by self-association of IgG-rheumatoid factors, since nearly all F(ab) fragments, prepared from the isolated complexes by pepsin digestion, bound to normal IgG. The association constants for the interaction between normal IgG and one binding site of the F(ab) fragments were about 10-5 liters/mole. Since a cyclic structure with two antigen-antibody bonds was thought to form in the self-association of two IgG-rheumatoid factors, the association constant for dimer formation was calculated to be 10-10 liters/mole. The preferential self-association of IgG-rheumatoid factor was supported by the observation that monomeric normal human IgG did not replace the IgG-rheumatoid factor when the complexes were dissociated and reformed in the presence of excess normal IgG. The self-association of IgG-rheumatoid factors may be a general phenomenon in rheumatoid arthritis, as suggested by the observations of other investigators.

Inactivation of Slow Reacting Substance of Anaphylaxis by Human Eosinophil Arylsulfatase

Arylsulfatase preferentially present in the human eosinophil as compared to other leukocytes was isolated by sequential gel filtration and cation exchange chromatography. The apparent molecular weight of 60,000, the preferential cleavage of 4-nitrocatechol sulfate (PNCS) over p-acetyl-benzenesulfonic acid (PABS), inhibition by phosphate ions and pH optimum of 5.7 are characteristics of a type II B arylsulfatase. Eosinophil arylsulfatase inactivated purified human slow reacting substance of anaphylaxis (SRS-A) in a time-dependent reaction with the rate dependent upon the enzyme/substrate ratio. That SRS-A inactivation was the result of intrinsic arylsulfatase activity was indicated by association of PNCS cleavage and SRS-A inactivating activity during chromatography, the similar pH optimum for cleavage of both substrates and the capacity of SRS-A to inhibit PNCS cleavage by arylsulfatase. The finding that eosinophil arylsulfatase inactivates SRS-A suggests that eosinophil ingress into the site of an immediate hypersensitivity reaction in response to ECF-A could represent a regulatory function.

Immunoglobulin origin of localized nodular pulmonary amyloidosis.

The major protein of an amyloid fibril concentrate from a patient with localized nodular pulmonary amyloidosis has been purified by sequential gel filtration on Sepharose 4 B and Sephadex G-100 columns using 5 M guanidine-HCl in 1 N acetic acid. The molecular weight of 15250, unreactive amino-terminal amino acid, peptide map pattern, and immunochemical cross-reactivity with some, but not all, lambda Bence Jones proteins identifies this amyloid fibril protein as being derived primarily from the aminoterminal segment of a homogeneous lambda light polypeptide chain of an immunoglobulin protein. Although the immunoglobulin origin of amyloid fibrils has been demonstrated in several pathologic settings, this is the first example of their immunoglobulin origin in a case of amyloidosis with restricted tissue involvement.

The purification of amyloid fibril proteins.

Abstract Amyloid fibril concentrates have been fractionated and shown to have homogeneous fragments of the variable region of immunoglobulin proteins as their major protein constituent. Amyloid fibril protein purification was performed on ten amyloid preparations by sequential gel filtration on Sepharose 4 B and Sephadex G-100 columns equilibrated with 5 M guanidine-HCl in 1 N acetic acid.

Amyloid Proteins: The Amino-Terminal Variable Fragment of Immunoglobulins

Abstract Purification of the major protein component of amyloid fibrils from human tissues and from tissues of the experimental murine disease has been accomplished by sequential gel filtration on Sepharose 4 B and Sephadex G-100 columns using 5 M guanidine-HCl in 1 N acetic acid with removal of over 28% of minor constituents. All amyloid proteins have a constant tryptophan content, a high dicarboxylic and short chain amino acid composition, an unreactive or Asx N-terminal group, monomeric molecular weight ranges of from 5000 to 18,300 and, in the human, heterogeneity of amyloid protein between individuals and homogeneity within the individual. Antibodies directed against the major amyloid protein of an individual cross-react with a component in the patient's serum. Immunologic evidence reveals the experimental murine protein to be derived from the N-terminal fragment of an immunoglobulin light chain of λ type and sequence analysis of a human protein categorizes it as derived from the N-terminal variable fragment of a κ I light chain.

Human amyloid protein: chemical variability and homogeneity.

The morphology of the fibril of amyloid derived from different individuals is similar, but occasionally significant differences are noted. All human amyloid filaments have a "β-pleated sheet" conformation as revealed by x-ray diffraction, and those examined after orientation show a "cross-β" pattern. All amyloid fibril concentrates studied so far can be fractionated to obtain the major amyloid protein component(s) by sequential gel filtration with 5 M guanidine-HCl in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns with the removal of over 28% of proteins representing minor constituents. The major amyloid protein(s) obtained from the spleen and/or liver of six patients is found to contain tryptophan, to be deficient in hydroxylysine and hydroxyproline and usually at least one commonly occurring amino acid and to have a high content of dicarboxylic acid and short chain amino acids and unreactive (blocked) NH2-terminal groups or aspartic acid-asparagine (Asx). However, the amyloid protein(s) from each individual differs from that of the others in molecular weight, in amino acid composition and in the presence or absence of specific tryptic peptides. Amyloid protein(s) from the liver and spleen of the same individual is identical. No chemical characteristics distinguish amyloid proteins derived from cases classified clinically as "primary" from those classified as "secondary." There is a striking chemical similarity between amyloid proteins and the NH2-terminal variable fragment of the light and heavy chain of immumoglobulin proteins.

Murine amyloid fibril protein: isolation, purification and characterization.

Murine amyloid has been produced by four different induction methods ( Mycobacterium butyricum, casein, casein plus Freund's adjuvant and endotoxin-induced mouse amyloidosis) in several strains and obtained from mice with "spontaneous" amyloidosis. The amyloid fibrils have been concentrated from spleen and liver. Electron microscopy of all of these preparations reveals the amyloid fibril to be 100 Å in width and composed of two parallel filaments, each measuring 35-40 Å in width and having the appearance of a twisted ribbon. X-ray diffraction of all preparations reveals a "backbone" spacing at 4.75 Å and a "side chain" spacing at 11 Å indicating a "β-pleated sheet" structure. Identification of the major protein component of amyloid fibril concentrates was made by combined use of sodium dodecyl sulfate polyacrylamide disc electrophoresis, labeling of the protein with 3H-tryptophan and Sepharose 4B gel filtration. Purification of the amyloid protein from spontaneous amyloidosis liver was accomplished by sequential gel filtration with 5 M guanidine in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns. The material is a unique glycoprotein with a molecular weight of 7200, a high content of dicarboxylic and short chain amino acids, a significant amount of tryptophan and an unreactive NH2-terminal amino acid, tentatively identified as pyrrolid-2-one-5-carboxylic acid. There are no methionine, half-cystine, hydroxylysine or hydroxyproline residues. Murine amyloid protein, therefore, has striking similarities to many human amyloid protein preparations. It differs from the human proteins in the similarity of molecular weights of different preparations.

Human amyloid protein: Diversity and uniformity

The major protein of amyloid fibril concentrates of ten tissues obtained from six patients has been fractionated by sequential gel filtration on Sepharose 4 B and Sephadex G-100 columns using 5 M guanidine-HCl in 1 N acetic acid with removal of over 28% of minor constituents. The major amyloid protein from each individual differs from that of the others in molecular weight, amino acid composition and peptide map profile. The major protein from the liver and spleen of the same individual, however, are identical. A serum component having an immunologic relationship to the patients' major amyloid protein has been detected migrating to the γ-globulin region on immunoelectrophoresis.