Polypeptide Fraction Research Articles

Biochemical variation between non-embryogenic and embryogenic calli of silver fir

Comparative study on SDS-protein profiles and isoenzyme composition of non-embryogenic and embryogenic calli in two callus lines of silver fir (Abies alba Mill.) revealed the presence of abundant polypeptide fractions and an increased number of isoperoxidases in non-embryogenic calli. Non-specific esterase, on the other hand, exhibited an opposite tendency, while glutamate dehydrogenase was the only enzyme system consisting uniformly of one isoenzyme band in both types of calli investigated.

APF/CBP, the small, amphipathic, anionic protein(s) in bile and gallstones, consists of lipid-binding and calcium-binding forms.

Two very similar small anionic, amphipathic proteins, a phospholipid-binding apoprotein (anionic polypeptide fraction [APF]) and a calcium-binding polypeptide (CBP), are found abundantly in bile and all types of gallstones. The often disparate properties among various preparations of APF/CBP could reflect different sources and separation procedures, leading to partly degraded and/or denatured protein and varied association of bile salts, lipids, bile pigments, and detergents. The present study presents new methods for isolation and purification of APF/CBP, and characterizes the preparations thus obtained. It was found that isolation by selective precipitation of proteins from fresh T-tube bile by added calcium chloride, followed by demineralization with ethylenediaminetetraacetic acid (EDTA), removal of salts, lipids, and some pigment by Sephadex LH-20, and serial ultrafiltration yields the purest preparations. Though free of lipids, bile salts, detergents, and most pigments, these new preparations all show the same 7-kd and 12-kd bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the same major peaks on hydrophobic high-performance liquid chromatography (HPLC), and retain the self-associative, lipid- and calcium-binding functions, typical of older preparations obtained by potentially denaturative procedures. The varied properties among APF/CBP preparations are thus apparently related mainly to their content of different proportions of two major components, lipid-binding APF and calcium-binding CBP. Immunologic cross-reactions indicate common epitopes, and amino acid analyses are also similar, suggesting that APF and CBP may have the same polypeptide backbone, but differ because of posttranslational modification(s). Sufficiently pure APF and CBP have now been obtained to permit possible structural identification by sequencing and molecular biological techniques, though such attempts have thus far been unsuccessful.

Uveitogenic 28/30 KD and 43 KD polypeptides in pigment epithelial membranes of the retina

The purpose of this study was the search for new intrinsic polypeptides of the retinal pigment epithelium (RPE) capable of evoking experimental uveitis. A membrane fraction was prepared from purified bovine RPE cells. The Triton X-100 soluble protein fraction was separated into polypeptide fractions by preparative gel electrophoresis, and the pathogenicity of the main isolated polypeptides was investigated in Lewis rats.After immunization, two hitherto unknown pigment epithelial polypeptides with Mr 28/30 KD (doublet) and 43 kD (PEP-28/30 and PEP-43, respectively) elicited progressive pigment epitheliitis and choroiditis accompanied by extending plaque-shaped macrophage accumulations along theRPE-Bruch's membrane layer. No inflammatory foci were found within the neural retina. Polypeptide fractions with Mr 14-17, 25 and 32/34 kD (doublet) (PEP-14/17, PEP-25 and PEP-32/34, respectively) appeared to be nonuveitogenic at the tested dose. PEP-28/30 and PEP-43 exhibited a partial antigenic relationship to PEP-65. PEP-28/30 exhibited marked reactivity to a monoclonal antibody previously raised to a 32 kD RPE-specific protein.In conclusion: in addition to the previously described main RPE-specific membrane polypeptide PEP-65, the RPE appears to contain two more uveitogenic components, the intrinsic pigment epithelial membrane polypeptides PEP-28/30 and PEP-43. Like PEP-65, these antigens are able to evoke experimental autoimmune pigment epithelial protein-induced uveitis (EAPU) in rats.

Biliary secretion of anionic polypeptide fraction is not coupled to that of phospholipids and cholesterol in rats.

Anionic polypeptide fraction (APF) is a phospholipid- and calcium-binding apoprotein present in animal and human bile, predominantly associated with cholesterol-phospholipid vesicles. In bile, the protein may play a physiological role in preventing precipitation of calcium salts. APF has also been suggested to be of regulatory importance in the process of biliary lipid secretion. The aim of the present study was to investigate whether the secretion rates of APF and that of biliary lipids are coupled, which would support a physiological role of APF in biliary lipid secretion. Biliary secretion rates of bile acids, phospholipids, and cholesterol were experimentally modulated in three different rat models. Secretion rates of APF were compared with that of bile acids, lipids, and with that of two other biliary proteins, the lysosomal protein beta-glucuronidase and apolipoprotein A-I (apo A-I). Model 1: diurnal variation in bile formation during chronic bile diversion; model 2: specific inhibition of biliary phospholipid and cholesterol, but not of bile acid secretion by infusion of the organic anion, sulfated lithocholyltaurine; model 3: acute interruption of the enterohepatic circulation in unanesthetized rats. The diurnal variation in bile formation involved a parallel increase of the biliary secretion rates of bile acids (+56 +/- 7%, mean +/- SD), phospholipids (+53 +/- 29%), cholesterol (+73 +/- 54%), and APF (+72 +/- 86%) during the night phase of the cycle. Infusion of sulfated lithocholyltaurine inhibited biliary phospholipid and cholesterol secretion (-78 +/- 15%, and -54 +/- 25%, respectively), but did not affect biliary bile acid or APF secretion rate (-19 +/- 14%, and +12 +/- 107%, respectively). Within 4 hours after interruption of the enterohepatic circulation, bile secretion rates for bile acids (-92 +/- 3%), phospholipids (-74 +/- 13%), cholesterol (-64 +/- 8%), and APF (-58 +/- 24%) rapidly declined to a new steady-state level. Correlation analysis using the data from the three experimental models indicated that the biliary secretion rate of APF was independent from that of phospholipids, cholesterol, beta-glucuronidase, and, presumably, apolipoprotein A-I, and positively correlated to bile acid secretion rate and bile flow. The data from three experimental models indicate that the biliary secretion rates of APF and of phospholipids/cholesterol are not coupled and, therefore, do not support a direct physiological role of APF secretion in biliary lipid secretion. APF secretion into bile may, at least partially, be controlled by biliary bile acid secretion.

Biliary secretion of anionic polypeptide fraction is not coupled to that of phospholipids and cholesterol in rats

Anionic polypeptide fraction (APF) is a phospholipid- and calcium-binding apoprotein present in animal and human bile, predominantly associated with cholesterol-phospholipid vesicles. In bile, the protein may play a physiological role in preventing precipitation of calcium salts. APF has also been suggested to be of regulatory importance in the process of biliary lipid secretion. The aim of the present study was to investigate whether the secretion rates of APF and that of biliary lipids are coupled, which would support a physiological role of APF in biliary lipid secretion. Biliary secretion rates of bile acids, phospholipids, and cholesterol were experimentally modulated in three different rat models. Secretion rates of APF were compared with that of bile acids, lipids, and with that of two other biliary proteins, the lysosomal protein β-glucuronidase and apolipoprotein A-I (apo A-I). Model 1: diurnal variation in bile formation during chronic bile diversion; model 2: specific inhibition of biliary phospholipid and cholesterol, but not of bile acid secretion by infusion of the organic anion, sulfated lithocholyltaurine; model 3: acute interruption of the enterohepatic circulation in unanesthetized rats. The diurnal variation in bile formation involved a parallel increase of the biliary secretion rates of bile acids (+56 +/- 7%, mean +/- SD), phospholipids (+53 +/- 29%), cholesterol (+73 +/- 54%), and APF (+72 +/- 86%) during the night phase of the cycle. Infusion of sulfated lithocholyltaurine inhibited biliary phospholipid and cholesterol secretion (-78 +/- 15%, and -54 +/- 25%, respectively), but did not affect biliary bile acid or APF secretion rate (-19 +/- 14%, and +12 +/- 107%, respectively). Within 4 hours after interruption of the enterohepatic circulation, bile secretion rates for bile acids (-92 +/- 3%), phospholipids (-74 +/- 13%), cholesterol (-64 +/- 8%), and APF (-58 +/- 24%) rapidly declined to a new steady-state level. Correlation analysis using the data from the three experimental models indicated that the biliary secretion rate of APF was independent from that of phospholipids, cholesterol, β-glucuronidase, and, presumably, apolipoprotein A-I, and positively correlated to bile acid secretion rate and bile flow. The data from three experimental models indicate that the biliary secretion rates of APF and of phospholipids/cholesterol are not coupled and, therefore, do not support a direct physiological role of APF secretion in biliary lipid secretion. APF secretion into bile may, at least partially, be controlled by biliary bile acid secretion.(Hepatology 1997 Jan;25(1):38-47)

STUDIES ON THE MECHANISM OF ACTION OF COPPER FINING AGENTS (κ CARRAGEENAN)

Carrageenan mediated clarification of brewers' wort comprises two reactions: interaction between carrageenan and soluble wort polypeptides and flocculation of particulate material. The dose/response characteristics of interaction between carrageenan and soluble polypeptide material suggested that carrageenan exhibits selectivity with respect to specific polypeptide fractions. Size exclusion chromatography of worts demonstrated that carrageenan treatment resulted in reductions in fractions of relative molecular mass Mr 70 000, 40 000 and 12 000. Fining trials demonstrated that the reactivity towards soluble polypeptides is independent of the presence of insoluble particulate material. Furthermore, both interaction with soluble polypeptides and flocculation of particulate material occurred in systems fined under high (80°C) and low (20–25°C) initial temperatures and this suggested that the presence of carrageenan in a helical conformation is essential to fining action rather than conformational transitions in polysaccharide structure. Additionally, carrageenan has no effect on the levels of low molecular mass wort solutes such as nucleic acid derivatives, aromatic and heterocyclic amino acids.

Rescue of Immunoglobulins from Insolubility Is Facilitated by PDI in the Baculovirus Expression System

A substantial fraction of immunoglobulin heavy and light chain polypeptides were insoluble when expressed in the baculovirus–insect cell expression system. In the presence of coexpressed heterologous protein disulfide isomerase (PDI), however, the solubility of the immunoglobulins was enhanced and IgG was secreted at higher levels from baculovirus-infectedTrichoplusia niinsect cells. Pulse-chase experiments indicated that some immunoglobulin polypeptides were initially insoluble in the presence of PDI but subsequently were rescued in a soluble form competent for IgG assembly and secretion. Recovery of the insoluble immunoglobulins was not observed in the absence of coexpressed PDI. Even after treatment of insect cells with tunicamycin to inhibit N-glycosylation of immunoglobulin heavy chains, coexpressed PDI was able to salvage insoluble immunoglobulins and secrete these modified glycoforms. The capacity for PDI to rescue immunoglobulins was also demonstratedin vitrowhere immunoglobulin heavy chains and light chain dimers were salvaged from aggregates of denatured IgG. PDI-mediated rescue of proteins, perhaps assisted by chaperones and other foldases, may be importantin vivowhere insolubility is a common occurrence for newly synthesized polypeptides.

Violaxanthin de-epoxidase.

Violaxanthin de-epoxidase catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Its activity is optimal at approximately pH 5.2 and requires ascorbate. In conjunction with the transthylakoid pH gradient, the formation of antheraxanthin and zeaxanthin reduces the photochemical efficiency of photosystem II by increasing the nonradiative (heat) dissipation of energy in the antennae. Previously, violaxanthin de-epoxidase had been partially purified. Here we report its purification from lettuce (Lactuca sativa var Romaine) to one major polypeptide fraction, detectable by two-dimensional isoelectic focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using anion-exchange chromatography on Mono Q and a novel lipid-affinity precipitation step with monogalactosyldiacylglyceride. The association of violaxanthin de-epoxidase and monogalactosyldiacyglyceride at pH 5.2 is apparently specific, since little enzyme was precipitated by eight other lipids tested. Violaxanthin de-epoxidase has an isoelectric point of 5.4 and an apparent molecular mass of 43 kD. Partial amino acid sequences of the N terminus and tryptic fragments are reported. The peptide sequences are unique in the GenBank data base and suggest that violaxanthin de-epoxidase is nuclear encoded, similar to other chloroplast proteins localized in the lumen.

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile: [formula omitted]. Dept of Gastroenterology, Tel-Aviv Medical Center and Tel-Aviv University, Israel and ∗INSERM U-130, Marseille, France

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile: [formula omitted]. Dept of Gastroenterology, Tel-Aviv Medical Center and Tel-Aviv University, Israel and ∗INSERM U-130, Marseille, France

Nitrocellulose immunoblotting for identification and molecular gene cloning of Eimeria maxima antigens that stimulate lymphocyte proliferation.

An immunoblotting technique was used to identify lymphostimulatory antigens within sized polypeptide fractions of Eimeria maxima sporozoites. Six fractions contained polypeptides that specifically stimulated the proliferation of immune lymphocytes in an in vitro assay, and polyclonal antisera were made in rabbits against these fractions. cDNA clones, isolated with antisera against a lymphostimulatory fraction of around 70 kDa, were found to encode four different antigens including a classical hsp70, a molecule homologous to an endoplasmic reticulum chaperonin (BiP/GRP), and a calcium-dependent serine/threonine protein kinase that appears homologous to a recently described molecule from Plasmodium falciparum. The protein kinase cDNA clone was overexpressed in Escherichia coli, and the recombinant antigen was found to induce both antibody and lymphoproliferative responses in chickens when administered subcutaneously. Thus, immunoblotting, in combination with in vitro lymphoproliferation assays, can be used as an initial screen for the identification of lymphostimulatory antigens from a complex pool of polypeptides, and a combination of cDNA cloning, expression, and immunization allows assessment of the lymphostimulatory activity of individual polypeptides. These studies should facilitate further evaluation of antigens that are potential candidates for inclusion in a recombinant vaccine against poultry coccidiosis.

Bile formation and hepatic plasma membrane composition in guinea-pigs and rats

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl −, phosphate and Ca 2+ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na +,K + ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs. Total fatty acid analysis of the membrane revealed that palmitic acid (C16:0), stearic acid (C18:0) and linoleic acid (C18:2, n-6) were the predominant fatty acids in guinea-pigs, while palmitic acid (C16:0), stearic acid (C18:0) and arachidonic acid (C20:4, n-6) were the most important in rats. Thus, high bile flow rate and BAIF in the guinea-pig may be attributed to the low bile acid concentration (below the critical micellar concentration), secretion of hypercholeretic bile acids (e.g. ketolithocholate) and high bicarbonate output.

Chaotrope-soluble cell wall glycoproteins of Volvox and some members of the Zygnematophyceae immunologically related to the ‘150 kDa’ cell wall glycoprotein of Chlamydomonas reinhardtii

As previously described, the cell wall of Chlamydomonas reinhardtii contains several chaotrope-soluble glycoproteins with similar sugar compositions, but considerably different proportions of hydroxyproline. All these hydroxyproline-containing cell wall glycoproteins are recognized by a polyclonal antibody raised against the purified ‘150 kDa’ cell wall glycoprotein of Chlamydomonas reinhardtii. This antibody also cross-reacts with some polypeptides present in the LiCl-extracts from intact cells of Spirotaenia erythrocephala, Spirotaenia obscura, Volvox aureus, and Volvox globator as revealed by Western blot analyses. Therefore, an IgG fraction of this particular antibody coupled to CNBr-activated Sepharose was used to isolate the immunologically related polypeptides extracted by aqueous LiCl from intact cells or cell wall preparations of different green algae belonging to the Volvocales or to the Zygnematophyceae. Different and more or less complex polypeptide patterns were observed by comparative SDS-PAGE analyses of the polypeptide fractions isolated by immunoaffinity chromatography from extracts of Chlamydomonas reinhardtii, Volvox aureus, Micrasterias rotata, Gonatozygon brebissonii and Netrium digitus, respectively, whereas the corresponding fraction prepared from Spirotaenia erythrocephala contained a predominant ‘160 kDa’ component. Amino acid and sugar analyses revealed that all these polypeptide fractions contained hydroxyproline (1.0–4.5 mol%) and the same sugars as the Chlamydomonas cell wall glycoproteins. However, the relative amounts of these sugars (arabinose, galactose, glucose, mannose and xylose) were found to be rather different. The highest proportion of hydroxyproline (4.5 mol%) and the highest ratio of arabinose: galactose (4.75:1) were determined for the glycoprotein fraction isolated from Spirotaenia erythrocephala indicating that at least the predominant ‘160 kDa’ component is an intrinsic cell wall glycoprotein containing hydroxyproline and oligo-arabinosyl side chains. All the polypeptide fractions isolated from the other green algae obviously also contained chaotropesoluble cell wall glycoproteins as revealed by the presence of arabinose and hydroxyproline. These findings show that all the investigated green algae contain chaotrope-soluble cell wall glycoproteins immunologically related to the ‘150 kDa’ cell wall glycoprotein of Chlamydomonas reinhardtii.

Short report: detection of 72-75-kD and 123-kD fractions of Leishmania antigen in urine of patients with visceral leishmaniasis.

Two polypeptide fractions of 72-75 kD were detected in the urine of 14 of 15 patients with visceral leishmaniasis (VL) and another fraction of 123 kD was found in 10 of the 15 patients by using a Western blot technique. None of these fractions was detected in the urine of 20 controls. These results suggest that antigen detection in urine could be a powerful, noninvasive method for VL diagnosis.

Purification of a bone sialoprotein-binding protein from Staphylococcus aureus.

Bone sialoprotein (BSP) is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone and joint tissues [Rydén C., Maxe, I., Franzén, A., Ljungh, A., Heinegård, D. & Rubin, K. (1987) Lancet II, 515]. We now report on the purification of a cell-wall protein from Staphylococcus aureus, strain O24, that possesses affinity for bone sialoprotein. Staphylococcal cell-wall components with capacity to inhibit binding of 125I-labeled BSP to staphylococcal cells were solubilized with LiCl (1.0 M, pH 5.0). Preparative SDS/PAGE and protein-overlay experiments revealed that inhibitory activity present in LiCl extracts resided in a fraction of polypeptides with M(r) 75,000-110,000. Staphylococcal proteins solubilized with LiCl were chromatographed on a Mono-Q anion-exchange column. Inhibitory activity was eluted at 0.6-0.8 M NaCl and could be further purified by affinity chromatography on BSP-Sepharose. Elution of the affinity matrix with 0.1 M glycine, pH 3.0, specifically eluted inhibitory activity. Analysis by SDS/PAGE revealed a single M(r) 97,000 polypeptide in the eluate. The purified M(r) 97,000 protein bound BSP in protein-overlay experiments. LiCl extracts from S. aureus, strain E514 or Staphylococcus epidermidis, strain 7686, both lacking the capacity to bind BSP did not contain the M9r) 97,000 protein. Our data demonstrate the presence of a S. aureus cell-surface BSP-binding protein. This protein could be involved in bacterial tropism in osteomyelitis.

Dissection of the Molecular Consequences of a Double Mutation Causing a Human Lysosomal Disease

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency in which leads to human storage disease aspartylglucosaminuria (AGU). AGUFin is the most common AGU mutation in the world and is found in 98% of AGU alleles in Finland, where the population displays enrichment of the disease allele. The AGUFin allele actually contains a double mutation, both individual mutations resulting in amino acid substitutions: Arg-161-->Gln and Cys-163-->Ser. The separate consequences of these two amino acid substitutions for the intracellular processing of the AGA polypeptides were analyzed using a stable expression of mutant polypeptides in Chinese hamster ovary (CHO) cells. The synthesized polypeptides were monitored by metabolic labeling, followed by immunoprecipitation, immunofluorescence, and immunoelectron microscopy. The Arg-161-->Gln substitution did not affect the intracellular processing or transport of AGA and the fully active enzyme was correctly targeted to lysosomes. The Cys-163-->Ser substitution prevented the early proteolytic cleavage required for the activation of the precursor AGA polypeptide and the inactive enzyme was accumulated in the endoplasmic reticulum (ER). The precursors of the translation products of the AGUFin double mutant and the Cys-163-->Ser mutant were also observed in the culture medium. When cells expressing the normal AGA or AGUFin double mutation were treated with DTT to prevent the formation of disulfide bonds, both normal and mutated AGA polypeptides remained in the inactive precursor form and were not secreted into the medium. These results indicate that correct initial folding is essential for the proteolytic activation of AGA.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of the biliary-anionic polypeptide fraction between cholesterol carriers in bile and its effect on nucleation

The small (7 kD) biliary phospholipid and calcium binding polypeptide (anionic polypeptide fraction/calcium binding protein) has been found in higher concentrations in the bile of patients with pigment stones than in controls. In different model systems it was variously found to promote or retard cholesteral crystalization. In the present study we investigated its distribution between cholesterol carriers in bile and its effect on cholesterol crystalization in native and model biles. On gel chromatography anionic polypeptide fraction/calcium binding protein was found predominantly in three areas: in the vesicular fraction, in the non-vesicular lipid fraction and in another fraction unassociated with biliary lipids. It was much more concentrated in the vesicular than in the non-vesicular fraction, the mean anionic polypeptide fraction/phospholipid molar ratio being 219 +/- 181 vs. 30.4 +/- 16, respectively. Anionic polypeptide fraction/calcium binding protein was added at three dose levels, 0.14, 0.28, 0.42 mg/ml (representing approximately 18%-55% of the physiologic biliary concentration), to 19 human and five model biles. This did not produce any significant changes in the nucleation time. The addition of anionic polypeptide fraction/calcium binding protein at a dose level of 0.42 mg/ml to 13 different human biles did not induce changes in the distribution of cholesterol among its carriers. The present experiments do not support a role for anionic polypeptide fraction/calcium binding protein in the process of cholesterol nucleation in bile. Qualitative changes in the protein molecule, as demonstrated in other human secretions, cannot be excluded.

Integrity of the 130 kda polypeptide of Bacillus Thuringiensis SSP. Israelensis δ-Endotoxin in K-S sporulation medium

The 130 kDa polypeptide appears to be the most susceptible of the polypeptides constituting the Bacillus thuringiensis ssp. israelensis δ-endotoxin to the proteases associated with sporulation. Stabilization of this polypeptide in the recently described K-S sporulation medium has been achieved through limiting its cleavage by removing contaminating proteases from the culture by washing. In this way, the complete δ-endotoxin profile is retained for studies on all of the constituent polypeptide fractions.

Anionic polypeptide fraction in bile of patients with and without gallstones

With the demonstration of pronucleating and antinucleating proteins, the role of biliary proteins became of considerable research interest. Anionic polypeptide fraction is the third most abundant biliary protein; it is found in association with biliary lipids, has antinucleating properties for calcium and is found in gallstones. Its levels in various human biles have not been studied as of this writing. In this investigation the concentration of anionic polypeptide fraction in gallbladder bile was measured in 16 subjects without gallstones, 19 subjects with cholesterol stones and 15 subjects with pigment stones in Tel Aviv. Anionic polypeptide fraction concentrations in bile (mean +/- S.D.) were 0.76 +/- 0.09 gm/L in controls and 0.81 +/- 0.25 gm/L (which was not significant) in patients with cholesterol gallstones. They were significantly higher 1.03 +/- 0.23 (p < 0.05) in patients with pigment gallstones. The anionic polypeptide fraction/phospholipid ratio and the anionic polypeptide fraction/total lipid ratio were significantly higher in patients with pigment gallstones (p < 0.005 and 0.05, respectively). The anionic polypeptide fraction lipid ratios were insignificantly elevated in biles of patients with cholesterol stones compared with the ratios in biles of controls. Only the anionic polypeptide fraction/phospholipid ratio was significantly higher in biles of patients with pigment stones compared with those with cholesterol gallstones. The values were similar although higher in a small group of gallstone patients from Marseilles. The role of anionic polypeptide fraction in the pathogenesis of gallstones, particularly pigment gallstones, requires further study.

Anionic polypeptide fraction in bile of patients with and without gallstones

Anionic polypeptide fraction in bile of patients with and without gallstones

High molecular weight proteins in the nematode C. elegans bind [3H]ryanodine and form a large conductance channel

Single-channel properties of a polypeptide fraction from the nematode Caenorhabditis elegans highly enriched in binding sites were studied in planar bilayers. [3H]Ryanodine binding sites were purified by sucrose gradient centrifugation of C. elegans microsomes solubilized in CHAPS detergent. The highest [3H]ryanodine binding activity sedimented at approximately 18% sucrose (wt/vol), and was composed of a major polypeptide with a M(r) of 360,000 and a minor polypeptide with a M(r) of 170,000. The ryanodine-binding polypeptide(s) formed a Ca(2+)-permeable channel with a permeability ratio P(divalent)/P(monovalent) = 4 and two conductance states of 215 pS and 78 pS in 0.25 M KCl. Ryanodine locked the channel in the 78 pS state and inhibited transitions between the 215 pS and 78 pS states. These data demonstrated the presence of a ryanodine receptor in C. elegans with functional properties comparable to those described in mammalian muscle.