Carboxymethyl Cellulose Column Research Articles

Studies of Bitter Peptides from Casein Hydrolyzate. I. Synthesis of Bitter Peptide BPIa Corresponding to Arg–Gly–Pro–Pro–Phe–Ile–Val from Casein Hydrolyzate by Alkaline Proteinase of Bacillus subtilis

Abstract A bitter heptapeptide BPIa was synthesized and compared with the natural peptide, isolated by Minamiura et al. from casein hydrolyzate, by means of thin layer chromatography, paper electrophoresis, and carboxymethylcellulose column chromatography. All results for the two peptides matched very closely each other. The synthetic BPIa has an extremely bitter taste with its threshold value 0.05 mM. It is one of the most bitter compounds such as phenylthiourea and quinine.

Detection of immunoreactive human placental oxytocin and its contractile effect on the uterine muscle.

Freshly obtained human placentas from various periods of gestation were quantitatively analysed for their immunoreactive oxytocin (OT) content and its biological activity was examined in a Magnus apparatus by utilizing rat uterus. The mean values for placental immunoreactive OT per gram tissue increased from the first to the second trimester, maintaining its high level to term. The total content of placental OT also increased continually from the beginning of pregnancy to term. Blood levels of estrogen stimulated neurophysin (ESN) and OT were concomitantly enhanced through gestation. Placental extract and synthetic OT showed similar peaks in the elution pattern of ion-exchange chromatography through a carboxymethyl cellulose column. Synthetic OT and placental extract induced marked uterine contraction in diestrous rats. However placental extract previously incubated with OT antiserum failed to induce this effect. Though detection of immunoreactive OT by immunoassay alone does not provide definite identification of pituitary and placental OT, the present study suggests that placental immunoreactive OT could have a contracting effect on the uterine muscle.

Separation of the subunits of crotoxin by high-performance liquid chromatography

Crotoxin is the major toxin in the venom of the Brazilian rattlesnake (Crotulus &%~sus terrificus)‘. It is a complex of two subunits associated non-covalently: a basic protein (crotoxin PLA or crotoxin B) which carries the phospholipase (PLA) activity and an acidic protein (crotapotin or crotoxin A) which has no enzymic activity and is not toxic2,3. The lethal effect of crotoxin has been attributed to a pre-synaptic blockage of the neuromuscular transmission 4, but it has been shown that the toxin acts also at the post-synaptic level 5,6. Habermann and Breithaupt’ stated that the separation of these two subunits was one of the three important goals in the study of crotoxin. In previous reports, these two subunits were separated after alkylation or acylation of the amino groups*, on a carboxymethylcellulose column at pH 4 or on a DEAE-cellulose column in 6 M urea 2,3,g. More recently, two subunits of human pituitary thyrotropin and pike eel gonadotropin have been successfully separated by hydrophobic interaction chromatography l”,ll. Judging from the amino acid compositions12, the two subunits of crotoxin have a large difference in hydrophobicity and thus may be separated by reversed-phase liquid chromatography. In this work the separation of the two subunits of crotoxin by high-performance liquid chromatography (HPLC) was investigated, and an unexpected finding is reported.

Characterization of mutagenic fractions in beef extract and in cooked ground beef. Use of blue-cotton for efficient extraction.

Mutagenic components in commercial beef extract and in cooked ground beef were adsorbed from their aqueous solutions on cotton bearing covalently linked trisulfo-copper-phthalocyanine residues (blue-cotton). By repeating the adsorption and elution, efficient concentration of the mutagenic components with a satisfactory overall recovery was achieved. Carboxymethyl cellulose column chromatography was found to be an excellent means to separate 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), two strong mutagens that have previously been found in heated beef samples. Chromatography of the mutagenic components of beef extract on this column gave two mutagenic fractions which corresponded to MeIQx and IQ in elution profile. In reversed-phase high performance liquid chromatography, the major active component of the MeIQx fraction and that of the IQ fraction behaved identically with standard samples of MeIQx and IQ, respectively. The contents of these mutagens in a sample of Difco beef extract were estimated at 200-300 ng of MeIQx and 20-40 ng of IQ per gram. By the same fractionation procedures, mutagenic substances in the cooked beef were fractionated into MeIQx-type and IQ-type components. The activity distribution among these two fractions was similar to that found for beef extract.

The primary structure of noxiustoxin: A K+ channel blocking peptide, purified from the venom of the scorpion Centruroides noxius Hoffmann

Noxiustoxin, component II-11 from the venom of scorpion Centruroides noxiusHoffmann, was obtained in pure form after fractionation by Sephadex G-50 chromatography followed by ion exchange separation on carboxy-methylcellulose columns (17). The primary structure of Noxiustoxin, a polypeptide 39 amino acid residues long was determined by automaticEdman degradation and chemical cleavage with cyanogen bromide followed by amino acid analysis of the two resulting peptides. Its sequence is: Thr−Ile−Ile−Asn−Val−Lys−Cys−Thr−Ser−Pro−Lys−Gln−Cys−Ser−Lys−Pro−Cys−Lys−Glu−Leu−Tyr−Gly−Ser−Ser−Ala−Gly−Ala−Lys−Cys−Met−Asn−Gly−Lys−Cys−Lys−Cys−Tyr−Asx−Asn, with a molecular weight of 4,184±6. No histidine, arginine, tryptophan or phenylalanine was found. Noxiustoxin is the first short toxin directed against mammals and the first K+ channel blocking polypeptide-toxin (4) found in scorpion venoms.

The purification and characterization of radiolabelled parathyroid hormone for radioimmunoassay

An improved method for the purification of 125I-labelled PTH is presented. Following iodination of the hormone by the chloramine-T method, the reaction mixture is transferred directly to a 0.9× 9 cm column of carboxymethyl-cellulose. Two major peaks emerge with the ammonium acetate gradient. Peak I represents labelled intact bPTH, M r 9600, of good quality, i.e. a high B 0 and a low NSB in the radioimmunoassay. Peak II, M r, approximately 12000–18000, gives a high B 0, but also a high NSB and is less suitable for RIA. Peak II is not the result of iodination damage, as it is also present in the unlabelled PTH (Inolex); it is suggested that it represents either a pre-prohormone or an aggregate of PTH. The label produced is usable for up to 3 months. Trasylol and benzamidine protect the label against proteolytic degradation equally well; the latter, however, is considerably cheaper.

Isoenzymes of myosin subfragments: Chromatographic fractionation on carboxymethylcellulose and actin-activated ATPase activity as a function of temperature

Soluble myosin fragments, subfragment-one (S-1) and heavy meromyosin, have been fractionated by chromatography on a column of carboxymethylcellulose into isoenzymes differing by their alkali light chains (A 1 and A 2). Both homodimers and the heterodimer of heavy meromyosin have thus been separated, as shown by polyacrylamide gel electrophoresis under dissociating and non-dissociating conditions. The Mg 2,+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activities of all these isoenzymes are similar in the absence of actin but, on the other hand, under the low ionic strength conditions of the experiments, significantly different in its presence. Actin activation has been more thoroughly studied in the case of the isoenzymes of S-1. It has been found, first, that the activation energies of the reactions catalyzed by acto-S-1 (A 1) and acto-S-1 (A 2) are not identical, and second, that, at 4°C, the maximal rate of ATP hydrolysis per mol actin at infinite S-1 (A 2) concentration is markedly higher than that expressed per mol S-1 (A 2) at infinite actin concentration; in the case of the reactions catalyzed by acto-S-1 (A 1), the turnover rates in both conditions of actin and enzyme excess are close. Analysis of these kinetics according to the currently accepted reaction schemes shows that the differences observed at low temperature between both isoenzymes reside in the last steps of the enzymatic reaction. These results suggest a possible distinct activity of the myosin heads in the cross-bridge cycle of muscular contraction.

Structural properties of Type I collagen isolated from chickens with scoliosis.

This study examines biochemically the Type I collagen isolated from skin of chickens that develop idiopathic scoliosis. Previous studies indicate a defect in collagen exists in these chickens. Alpha 1 (I) and alpha 2 chains were separated by gel filtration and carboxymethyl cellulose column chromatography and were then subjected to the analytical techniques of sodium dodecyl sulfate gel electrophoresis, Staphylococcus aureus V8 protease digestion, cyanogen bromide peptide mapping and amino acid analyses. In all categories, the scoliotic alpha 1 (I) and alpha 2 chains were identical to alpha chains isolated from normal chickens. These data suggest that the altered properties of collagen solubility and connective tissue stress relaxation seen in these scoliotic chickens are not a manifestation of an altered primary structure of the alpha chains or post-translational modification affecting chromatographic elution profiles or electrophoretic migration patterns.

6] Arachidonic acid-15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes

This chapter presents the procedure for purification and assay of arachidonic acid-15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. All operations in purification were done at 0–4 °, crude extraction cells were sonicated 15 times for 30 sec in 60 ml of 50mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. Further procedure steps are (1) acetone fractionation, (2) carboxymethyl-cellulose column chromatography, (3) gel filtration on sephadex G-150, and (4) hydroxyapatite column chromatography. The enzyme was eluted at approximately 350 mM buffer concentration. The active fractions were pooled and concentrated by ultrafiltration to about 1 ml. The activity of arachidonic acid-15-1ipoxygenase was assayed by measuring the sum of the radioactivities converted from [1-14C]arachidonic acid to 15-hydroperoxyeicosatetraenoic and its decomposition products. The radioactivity was determined in a scintillation counter. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the formation of 1μmol of product in 2 min at 30 °.

Protection of rat liver 80 S ribosomes against ricin A chain inactivation by proteins extracted from rat liver and wheat germ ribosomal subunits with [formula omitted

Proteins extracted from wheat germ 60 S ribosomal subunits and rat liver 60 S and 40 S ribosomal subunits with 3 M NH 4 Cl 75 mM MgCl 2 were able to prevent the ricin A chain-mediated inactivation of untreated 80 S rat liver ribosomes. The protection of polyphenylalanine synthetic capability of 80 S ribosomes was saturable and reached 100% protection in the presence of about 20 μg of extracted protein using a uniform set of assay conditions. No protection was observed using proteins extracted from wheat germ 40 S subunits or the core fraction of rat liver 60 S subunits or protein extracted from Escherichia coli ribosomes or ribosomal subunits. The conclusion that the protective effect of extracted 60 S subunit proteins was specific, was further strengthened by showing that unrelated proteins such as α-lactalbumin, bovine serum albumin and lysozyme, and polypeptides such as polylysine and poly(aspartic acid), also showed no protection. If 80 S ribosomes were first treated with ricin A chain and then incubated with proteins extracted from rat liver 60 S subunits, no protection was observed. Proteins extracted with NH 4 Cl MgCl 2 from 60 S rat liver subunits were applied to a carboxymethylcellulose column equilibrated with 6 M urea. Stepwise elution with increasing concentrations of LiCl resulted in seven fractions. One fraction (D) contained most of the protective factor; one fraction (E) contained a lesser amount of the protective factor. Two-dimensional polyacrylamide gel electrophoresis of fraction D showed the presence of ten proteins. These data are consistent with the idea that the enzymatic target of ricin A chain is protein is nature and that fraction D contains one or more proteins that appear to act as a inhibitor against ricin A chain.

Regeneration of ribonuclease A from the reduced protein. Isolation and identification of intermediates, and equilibrium treatment.

Reduced RNase A was reoxidized, and the incorrectly formed disulfide bonds were reshuffled to the native ones by oxidized and reduced glutathiones, as described in the first paper of this series. The intermediates in the regeneration of the disulfide bonds were trapped without any chemical modification and were fractionated on a carboxymethylcellulose column at pH 3.5 with a salt gradient. The elution curves of the partially regenerated RNase A from the carboxymethylcellulose column were obtained by measurement of the absorption at 275 nm and by determination of the SH content (of cysteine residues) and consisted of 11 fractions, G8, G7, G6, G5, G4, G3, G2, G1, G0, N, and F. Some of the fractions were isolated, and their measured molecular weights were consistent with those of monomeric RNase A. Fraction F had a molecular weight between that of the monomer and dimer, so that this fraction could not be identified. The regeneration pathway could be represented in terms of two simple reactions, RNase A(-SH) + GSSG in equilibrium or formed from RNase A(-SSG) + GSH and RNase A(-SH-SSG) in equilibrium RNase A(greater than S2) + GSH, which produced 24 monomeric intermediates (not counting the fully reduced and the native species), which differed from each other in their amino acid composition. These 24 intermediates, plus the fully reduced protein, were assigned to fractions G8--G0 (as indicated in the last column of Table I), with the aid of data from amino acid analysis, SH content, and the elution position on the carboxymethylcellulose column chromatogram. Since the regeneration reaction rapidly reached a preequilibrium among the intermediates and the fully reduced RNase A prior to the rate-limiting steps, i.e., the relative concentrations of the intermediates and fully reduced RNase A became constant with reaction time, the populations of some of the intermediates in preequilibrium were estimated by curve fitting of the elution pattern from the carboxymethylcellulose column chromatogram. The equilibrium constants among the intermediates were calculated from their populations at preequilibrium. These equilibrium constants were "extrapolated" to other intermediates whose populations could not be estimated by curve fitting, and the relative populations of all of the possible intermediates at preequilibrium were thereby represented as a function of the concentrations of reduced and oxidized glutathiones. The regeneration process was also restarted from several of the isolated intermediates, and the resulting distribution of intermediates was consistent with that from which the equilibrium constants were determined, supporting the representation of the regeneration pathways in terms of two simple reactions. Thus, the equilibrium treatment of the regeneration pathways was useful to characterize the preequilibrium state, i.e., to identify the intermediates prior to the rate-limiting steps in the pathways and to estimate their stabilities at preequilibrium at various concentrations of reduced and oxidized glutathiones.

Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly.

The 200,000-dalton neurofilament subunit (P200) and the 160,000-dalton (P160) and 78,000-dalton (P78) neurofilament subunits were partially purified from bovine brain. Intact neurofilaments were prepared by high-speed and sucrose-zone centrifugation. The crude neurofilament was solubilized in 8 M urea solution containing pyridine, formic acid, and 2-mercaptoethanol. The solubilized neurofilament was purified by carboxymethyl (CM) cellulose column and hydroxylapatite column chromatography. The P200 was purified as separate from P160 and P78, but the P160 and P78 subunits were copurified on CM cellulose, hydroxylapatite, Bio-Gel A150m, and Sephadex G-150 column chromatography. Electron microscopy of these purified neurofilament subunits revealed the P200 subunit as a globular structure, and the P160 and P78 subunits as a rod-shaped structure extending up to 120 nm with a 8- to 12-nm width. In the presence of 200 mM KCl, 15 mM MgCl2, and 1 mM ATP, the purified subunits assembled into long filaments. Under the assembly condition, P160 and P78 subunits elongated up to 500 nm, but the longer filament formation required the presence of P200 subunits. The filaments formed in vitro were of two types: long straight filaments and intertwined knobby-type filaments. From these results, we have suggested that P160 and P78 form the neurofilament backbone structure and P200 facilitates the assembly of the backbone units into longer filaments.

Hemoglobin Petah Tikva (a 110 Ala → Asp): A New Unstable Variant With α-Thalassemia-Like Expression

AbstractHemoglobin Petah Tikva, a new unstable α-Chain structural variant, was identified in two unrelated children whose families are from the Iraqi Jewish community of Baghdad. Both children have α-thalassemia. with hematologic features of mild HbH disease. The abnormal hemoglobin from the two patients did not separate from hemoglobin A by electrophoresis or chromatography, but the mutant α-chain emerged ahead of αA in carboxymethylcellulose column chromatography, allowing it to be isolated in pure form. Hemoglobin Petah Tikva was unstable in solution and could also be isolated by isopropanol precipitation. Structural analyses indicate that the variant α-Chains contain a normal number of amino acids and have a substitution of Ala → Asp at α 110. Each of the patients with HbH disease has one parent with hematologic findings and globin chain synthesis ratios that are characteristic of α-thalassemia trait, and a hematologically normal parent who is a carrier of the variant α-chain of Hb Petah Tikva. The patients each appear to be doubly heterozygous for α-thal-1 and for the structurally abnormal hemoglobin. The mutant α-chains of Hb Petah Tikva were synthesized by reticulocytes of the heterozygous carriers, but only traces of αPT globin could be detected in their blood, suggesting that the abnormal α-chains underwent postsynthetic degradation or precipitation in these individuals. In the patients with Hb Petah Tikva in combination with α-thalassemia, the normal and mutant α-chains were synthesized by bone marrow erythroid cells at rates proportional to their concentrations in the blood, but there was a substantially decreased rate of synthesis of the variant α-chain by their blood reticulocytes. The α-thalassemia-like expression of Hb Petah Tikva appears to result from instability of the abnormal α-chain as well as from the premature termination of its synthesis during erythroid cell maturation.

Purification and properties of endo-alpha-1,3-glucanase from a Streptomyces chartreusis strain.

An enzyme hydrolyzing the water-insoluble glucans produced from sucrose by Streptococcus mutans was purified from the culture concentrate of Streptomyces chartreusis strain F2 by ion-exchange chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose columns and gel filtration on Bio-Gel A-1.5m. The purification achieved was 6.4-fold, with an overall yield of 27.3%. Electrophoresis of the purified enzyme protein gave a single band on a sodium dodecyl sulfate-polyacrylamide gel slab. Its molecular weight was estimated to be approximately 68,000, but there is a possibility that the native enzyme exists in an aggregated form or is an oligomer of the peptide subunits, have a molecular weight larger than 300,000. The pH optimum of the enzyme was 5.5 to 6.0, and its temperature optimum was 55 degrees C. The enzyme lost activity on heating at 65 degrees C for 10 min. The enzyme activity was completely inhibited by the presence of 1 mM Mn2+, Hg2+, Cu2+, Ag2+, or Merthiolate. The Km value for the water-insoluble glucan of S. mutans OMZ176 was an amount of glucan equivalent to 1.54 mM glucose, i.e., 0.89 mM in terms of the alpha-1,3-linked glucose residue. The purified enzyme was specific for glucans containing an alpha-1,3-glucosidic linkage as the major bond. The enzyme hydrolyzed the S. mutans water-insoluble glucans endolytically, and the products were oligosaccharides. These results indicate that the enzyme elaborated by S. chartreusis strain F2 is an endo-alpha-1,3-glucanase (EC 3.2.1.59).

Isolation and partial amino acid sequence analysis of nerve growth factor from the guinea pig prostate

Nerve growth factor (NGF) has been purified from the guinea pig prostate using a modification of the Bocchini-Angeletti method for isolating 2.5S NGF from mouse submaxillary glands. As with the mouse preparation, guinea pig prostate NGF appears to migrate as a high molecular weight entity at physiological pH. Following dissociation, NGF, active in neurite proliferation assays and similar in size to mouse 2.5S NGF, can be isolated by chromatography on a column of carboxymethyl-cellulose at pH 4.8. Based on gel filtration, SDS-polyacrylamide gel analysis, and amino-terminal sequence studies, this material consists of two, noncovalently linked, identical polypeptide chains each with a molecule weight of about 13,000. The amino-terminal third of the polypeptide chain is at 90% identical to the corresponding region of the murine molecule, confirming the homology of the guinea pig prostate protein to NGFs obtained from different tissues in other species. However, in contrast to the mouse preparation, the putative high molecular weight form of guinea pig NGF does not contain a subunit with arginine esteropeptidase activity. Although there is an abundance of this enzymatic activity in the homogenate, it does not appear to be associated with the fractions containing NGF. This apparent difference in the mouse and guinea pig material is of interest because the mouse gamma subunit, possessing the arginine esteropeptidase activity, has been alleged to participate in the processing of a precursor of the beta NGF polypeptide chain.

42] Cathepsin G

This chapter presents the procedure for purification and assaying of cathepsin G. Cathepsin G is purified from human neutrophil leukocytes. Cathepsin G is eluted from the carboxymethyl-cellulose column at a higher salt concentration than the elastase. The cathepsin G tends to be contaminated by traces of elastase, which are readily removed by immunoadsorption, or by rechromatography on carboxymethyl-cellulose. Cathepsin G is well stored by lyophilization from solution in a 0.5 M pyridine acetate buffer, pH 5.5. Solutions in 0.5 M NaC1 are also stable at 20°C for long periods. The activity of cathepsin G can be detected in electrophoresis gels by use of a naphthol ester substrate such as Bz-Phe-ONap or the acetate or chloroacetate of naphthol AS-D, liberated naphthol being coupled with a diazonium salt. Moreover, it is quite possible that a burst-assay with a nitrophenyl ester could be adapted for cathepsin G.

Biochemical characterization of a toxin from indian cobra ( Naja naja naja) venom

A major toxic component was isolated from the venom of Indian cobra ( Naja naja naja) by ammonium sulfate fractional precipitation followed by carboxymethyl cellulose column chromatography and Sephadex gel filtration. This component constituted 2% of the venom and produced a block of neuromuscular transmission in nerve muscle preparations. Three other toxic fractions comprising 3% of the venom were also detected. The major toxic component was homogeneous on starch and polyacrylamide gel electrophoresis and on rechromatography on CM-cellulose. Its molecular weight was approximately 6300. This toxin contained 61 amino acid residues including 8 half-cystine residues whereas alanine, methionine and phenylalanine were totally absent. Its ld 50, as determined by i.p. injection in mice, was 0·2 mg per kg body weight. The fraction did not possess any enzymatic, hemolytic or hemagglutinin activities of crude venom but showed a close resemblance to the major neurotoxin of Formosan cobra venom.

Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11.

The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. D., & Fraser, W. (1980) Biochemistry 19, 1425-1433]. Three relatively acidic proteins, S14, S25/S27, and 7/8, have now been isolated from group A80 by ion-exchange chromatog raphy on carboxymethylcellulose eluted with a linear gradient of lithium chloride at pH 4.2. Fractions containing the relatively basic proteins (groups B80 through G80) were furher combined into a total of 24 "pools". The criterion for combination was the migration patterns in one-dimensional polyacrylamide gels containing sodium dodecyl sulfate (NaDodS04) of every fifth fraction from the carboxymethylcellulose column. Each pool contained between 1 and 12 major proteins. Proteins S8, S13, S16, S19, S20/L24, S22/L26, S24, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 9, and 11 have now been isolated from selected pools by gel filtration through Sephadix G-100. The amount of each protein recovered from a starting amount of 1.8 g of total 80S proteins varied form 0.2 to 10.8 mg. Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.

Analytical chromatography of ribosomal proteins in the fungus, Podospora anserina

The structure of ribosomes is extremely complex. Both genetical and biochemical analyses are necessary to understand the relationships between their structure and their functions. In procaryotes, the involvement of ribosomal proteins in different functions such as resistance to some antibiotics (see for review (1)) or control of translational fidelity (see for review (2)) has been demonstrated by isolation of mutants modified for these functions and identification of an altered ribosomal protein. Two methods have been used to compare the ribosomal protein patterns of two strains: 1. (a) electrophoretic analysis: essentially two-dimensional electrophoresis on polyacrylamide gels (3). 2. (b) cochromatography of differentially labeled ribosomal proteins from the two strains on a column of carboxymethylcellulose (CM-cellulose) (4). This latter method was often more reliable and more highly resolving than the former method (5,6). Such studies are not so advanced in eukaryotes with respect to cytoplasmic ribosomes. Most of the ribosomal mutants analyzed had unaltered electrophoretic profiles of ribosomal proteins and have not been analyzed further using other methods. Identification of an altered protein in cytoplasmic ribosomes has been reported in two fungi (7,8) and in mammalian cells (9) for mutants resistant to antibiotics. For our part we possess a large number of ribosomal mutants involved in the control of translational fidelity in a fungus, Podospora anserina (10–12). Up to now the electrophoretic analysis has been disappointing. Therefore we have adapted CM-cellulose chromatography for the comparison of ribosomal proteins from wild-type and mutant strains of Podospora. This method of analytical chromatography is presented in this paper.

Characterization of collagens of diseased human gingiva.

In the gingiva and other connective tissues, alteration in the collagens is primarily responsible for their functional impairment during disease. To study the collagen alterations, we extracted diseased human gingival tissue with neutral and acidic solvents and then with pepsin. The pepsin extract was separated into proteins soluble in 2.5 and 1.5 M NaCl and proteins insoluble in 1.5 M NaCl. By the criteria of solubility behavior in NaCl solutions, elution from (carboxymethyl)cellulose (CM-cellulose) columns, sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis, CNBr peptide pattern, and amino acid composition, the collagens of acidic and neutral solvent extracts and 1.5 M soluble fraction of pepsin extract were characterized as type I collagen and the 1.5 M NaCl insoluble collagen as type III. The 2.5 M NaCl fraction contained alpha 2, A, and B chains. The alpha 1 chains resembled alpha 1[I] in amino acid composition, and, since alpha 2 chains were lacking, it appeared that these chains derived from type I trimer collagen. The A and B chains were purified from the 2.5 M NaCl fractions by salting out at acidic pH. The final (A plus B) chain fraction was resolved into two major and one minor protein peaks by phosphocellulose chromatography. The major peaks were characterized as A and B chains on the basis of amino acid composition and CNBr peptide patterns. The minor peak had electrophoretic mobility slightly less than B chains, and the amino acid composition was different. Analysis of the proportion of different collagen types extracted indicated that type III collagen, which is the second major fraction in other connective tissues, is only a minor constituent in the gingiva. More interestingly, A and B chains accounted for a greater proportion than type III. Unlike the fibroblast cultures, the type I trimer formed only a small proportion of collagens of diseased gingival tissue.