Carnitine palmitoyltransferases in pea leaf chloroplasts: partial purification, location, and properties

2000 ◽  
Vol 78 (3) ◽  
pp. 328-335 ◽  
Author(s):  
Christine Masterson ◽  
Clifford Wood
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Carnitine Palmitoyltransferase ◽  
Chloroplast Envelope ◽  
Partial Purification ◽  
Western Blots ◽  
Chain Acyl ◽  
Long Chain ◽  
Inner Envelope

Carnitine palmitoyltransferase (EC 2.3.1.21), an enzyme that catalyses the reversible transfer of activated long-chain acyl groups between CoASH and L-carnitine, has been confirmed in pea leaf chloroplasts. This enzyme is bound to the chloroplast inner envelope membrane and has two isoforms, one bound to the outside (cytosol side) of the inner envelope and one bound to the inside (stromal side) of the inner envelope. Malonyl CoA inhibited the activity of the outer carnitine palmitoyltransferase, while stimulating the activity of the inner isoform and may be a regulator of these enzymes in vivo. Carnitine palmitoyltransferase was solubilized from the chloroplast envelope by detergent treatment and the two isoforms separated by Q-Sepharose anion exchange chromatography. Both proteins were immunochemically observed by probing Western blots of sodium dodecyl sulfate - polyacrylamide gel electrophoresis gels using an anti-beef heart mitochondrial carnitine palmitoyltransferase polyclonal antibody. The monomeric molecular mass of the protein recognized by this antibody was approximately 20 kDa. This 20-kDa protein also bound3H-carnitine. Both isoforms had broad acyl CoA substrate specificities, but showed increased activity with desaturated long-chain acyl CoAs, exhibiting a preference for linolenoyl CoA. A role for carnitine palmitoyltransferase in the shuttling of fatty acids across the chloroplast envelope is suggested.Key words: Pisum sativum, chloroplasts, carnitine palmitoyltransferase, fatty acid metabolism, eukaryotic pathway, membrane transport.

scholarly journals Variable major proteins of Borrellia hermsii.

1982 ◽  
Vol 156 (5) ◽  
pp. 1312-1324 ◽  
Author(s):  
A G Barbour ◽  
S L Tessier ◽  
H G Stoenner
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Antigenic Variation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Relapsing Fever ◽  
Western Blots

Borrelia hermsii, a relapsing fever agent, manifests antigenic variation in vivo and in vitro. We studied three mouse-passaged serotypes of strain HS1 (7, 14, and 21) and a HS1 derivative obtained after multiple in vitro passages (C serotype). All four serotypes had two major proteins in whole cell lysates fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One major protein species (pII) had the same apparent subunit molecular weight (or approximately 3.9 X 10(4) in all the serotypes. In contrast, the other abundant protein in lysates, pI, had a different apparent molecular weight in each serotype. In one gel the molecular weights of pIc, pI7, pI14, and pI21 were 1.9, 4.2, 4.1, and 4.0 X 10(4), respectively. Serotype-specific mouse antisera bound to both hemologous and heterologous pIIs, to homologous pI, but not to heterologous pI in Western blots. Hybridomas were raised from spleens of mice infected with B. hermsii. Monoclonal antibodies were identified by immunofluorescence assays using whole organisms. Monoclonal antibodies specific for serotype 7 (H1826) or for serotype 21 (H3326) bound only to pI7 or pI21, respectively, in Western blots. The surface location of the pI was suggested not only by the immunofluorescence studies but also by the labeling of pI7 and pI21 when whole cells of serotypes 7 and 21 were incubated with 125I in the presence of Iodogen. Under the same circumstances, pII was relatively poorly labeled. These studies have identified the variable pI proteins of B. hermsii as serotype-specific antigens. A change from one pI to another may be the basis of antigenic variation of Borrelia species during relapsing fever.

Schistosoma mansoni: anomalous immunogenic properties of a 27 kDa larval serine protease associated with protective immunity

Parasitology ◽  
1997 ◽  
Vol 115 (3) ◽  
pp. 237-247 ◽  
Author(s):  
H. Y. DARANI ◽  
R. H. C. CURTIS ◽  
C. McNEICE ◽  
H. P. PRICE ◽  
J. R. SAYERS ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Serine Protease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Molecular Size ◽  
Major Constituent ◽  
Partial Purification ◽  
Western Blots ◽  
Sds Page

A cationic Schistosoma mansoni cercarial antigen was shown to be a serine protease as it was capable of hydrolysing N-acetyl-dl-phenylalanine β-naphthyl ester (NAPBNE) after precipitation by immunoelectrophoresis, and this reaction was modulated by the serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP). The antigen in the immunoprecipitin arcs could also be radio-isotope labelled with tritiated DFP. The peptidolytic enzyme identified in immunoelectrophoresis with polyspecific sera and radio-isotope labelled with tritiated DFP had a relative molecular size of approximately 27 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), and evidence obtained after partial purification, SDS–PAGE and immunoblotting supported this size estimate for the enzyme. A rabbit antiserum raised against the peptidolytic antigen reacted against a doublet of antigens at 27/28 kDa in immunoelectrophoresis arcs and against an antigen of 60 kDa in Western immunoblots of crude cercarial homogenate. However, the latter serum precipitated the cationic antigen in immunoelectrophoresed cercarial homogenates only after pre-incubation of the homogenates with PMSF. Fractions containing the partially purified protease also degraded radio-isotope labelled human IgG. The reactivity of a range of polyspecific and monospecific rabbit antisera in Western blots with larval extracts indicated that antibody responses against the 27/28 kDa doublet may be modulated. When immunized with material which contained the 27 kDa enzyme as a major constituent, and which was secreted by S. mansoni cercariae during transformation, only 5 of 16 mice produced antibody to this antigen that was detectable in Western blots. The 5 antibody ‘responder’ mice were significantly (P<0·001) protected against challenge with a percutaneous infection of S. mansoni cercariae compared with a group of mice also immunized with CTF, but which had not produced antibodies against the 27/28 kDa doublet. The results indicate that the 27 kDa serine protease of S. mansoni larvae is a target that is sensitive to immunological attack.

Eimeria tenella sporozoites: the method of excystation affects the surface membrane proteins

Parasitology ◽  
1987 ◽  
Vol 95 (3) ◽  
pp. 479-489 ◽  
Author(s):  
M. H. Wisher ◽  
M. E. Rose
Keyword(s):  
Bile Salts ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Eimeria Tenella ◽  
Immunofluorescent Staining ◽  
Antigenic Determinants ◽  
Western Blots

SUMMARYEimerian sporozoites can be recovered from intestinal washings after oral administration of oocysts to chickens but suspensions of sporozoites are usually prepared in the laboratory by incubation of sporocysts or fractured oocysts in vitro, at body temperatures, with relatively high concentrations of trypsin and bile salts. Since these agents affect membrane structure, the surface membrane of proteins of Eimeria tenella sporozoites excysted in vivo and in vitro have been compared. Surface radio-iodination followed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) showed that more 125I was incorporated into polypeptides on sporozoites excysted in vivo than on sporozoites excysted in vitro. The 125I-polypeptide profile of sporozoites excysted in vivo was more resistant to subsequent incubation with pure trypsin than that of sporozoites excysted in vitro, but incubation with bile salts resulted in the loss of some iodinated polypeptides from both preparations of iodinated sporozoites. Reaction with combinations of crude trypsin and bile salts led to the lysis of sporozoites. The method of excystation had no effect on the reaction of convalescent chicken serum with Western blots of sporozoites but the results of immunofluorescent staining carried out with mouse monoclonal antibodies indicated that the structure of the cell surface was altered and some antigenic determinants were lost from sporozoites excysted in vitro. In contrast, neither the infectivity of sporozoites determined in vivo, nor their invasion of cultured cells was changed by the method of excystation.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

scholarly journals Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell

1984 ◽  
Vol 220 (2) ◽  
pp. 371-376 ◽  
Author(s):  
S Soboll ◽  
H J Seitz ◽  
H Sies ◽  
B Ziegler ◽  
R Scholz
Keyword(s):  
Liver Cell ◽  
Adenine Nucleotide ◽  
Intact Cell ◽  
Inhibitory Effect ◽  
Fatty Acyl ◽  
Fed State ◽  
Physiological Relevance ◽  
Chain Acyl ◽  
Long Chain

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

scholarly journals The interrelations between high- and low-molecular-weight forms of normal and mutant (Krabbe-disease) galactocerebrosidase

1980 ◽  
Vol 189 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Yoav Ben-Yoseph ◽  
Melinda Hungerford ◽  
Henry L. Nadler
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Krabbe Disease ◽  
Low Molecular Weight ◽  
Molecular Weight Form

Galactocerebrosidase (β-d-galactosyl-N-acylsphingosine galactohydrolase; EC 3.2.1.46) activity of brain and liver preparations from normal individuals and patients with Krabbe disease (globoid-cell leukodystrophy) have been separated by gel filtration into four different molecular-weight forms. The apparent mol.wts. were 760000±34000 and 121000±10000 for the high- and low-molecular-weight forms (peaks I and IV respectively) and 499000±22000 (mean±s.d.) and 256000±12000 for the intermediate forms (peaks II and III respectively). On examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the high- and low-molecular-weight forms revealed a single protein band with a similar mobility corresponding to a mol.wt. of about 125000. Antigenic identity was demonstrated between the various molecular-weight forms of the normal and the mutant galactocerebrosidases by using antisera against either the high- or the low-molecular-weight enzymes. The high-molecular-weight form of galactocerebrosidase was found to possess higher specific activity toward natural substrates when compared with the low-molecular-weight form. It is suggested that the high-molecular-weight enzyme is the active form in vivo and an aggregation process that proceeds from a monomer (mol.wt. approx. 125000) to a dimer (mol.wt. approx. 250000) and from the dimer to either a tetramer (mol.wt. approx. 500000) or a hexamer (mol.wt. approx. 750000) takes place in normal as well as in Krabbe-disease tissues.

Isolation, Cultivation, and Characterization of Borrelia burgdorferi from Rodents and Ticks in the Charleston Area of South Carolina

2000 ◽  
Vol 38 (1) ◽  
pp. 120-124
Author(s):  
J. H. Oliver ◽  
K. L. Clark ◽  
F. W. Chandler ◽  
L. Tao ◽  
A. M. James ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Borrelia Burgdorferi ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Cotton Rat ◽  
Cotton Mouse ◽  
B 31

ABSTRACT Twenty-eight Borrelia burgdorferi isolates from the Charleston, S.C., area are described. This represents the first report and characterization of the Lyme disease spirochete from that state. The isolates were obtained from December 1994 through December 1995 from the tick Ixodes scapularis , collected from vegetation, and from the rodents Peromyscus gossypinus (cotton mouse), Neotoma floridana (eastern wood rat), and Sigmodon hispidus (cotton rat). All isolates were screened immunologically by indirect immunofluorescence with monoclonal antibodies to B. burgdorferi -specific outer surface protein A (OspA) (antibodies H5332 and H3TS) and B. burgdorferi -specific OspB (antibodies H6831 and H614), a Borrelia (genus)-specific antiflagellin antibody (H9724), Borrelia hermsii -specific antibodies (H9826 and H4825), and two polyclonal antibodies (one to Borrelia species and another to B. burgdorferi ). Six of the isolates were analyzed by exposing Western blots to monoclonal antibodies H5332, H3TS, H6831, and H9724. All isolates were also analyzed by PCR with five pairs of primers known to amplify selected DNA target sequences specifically reported to be present in the reference strain, B. burgdorferi B-31. The protein profiles of six of the isolates (two from ticks, one from a cotton mouse, two from wood rats, and one from a cotton rat) also were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the 28 Charleston isolates are B. burgdorferi sensu stricto based on their similarities to the B. burgdorferi B-31 reference strain.

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