scholarly journals Purification of non-histone acceptor proteins for ADP-ribose from mouse testis nuclei

1984 ◽  
Vol 221 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M R Faraone Mennella ◽  
P Quesada ◽  
B Farina ◽  
E Leone ◽  
R Jones
Keyword(s):  
Average Length ◽  
Specific Radioactivity ◽  
High Mobility ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Mouse Testis ◽  
Histone Proteins ◽  
High Mobility Group Proteins ◽  
Associated Proteins

Acceptor proteins for poly(ADP-ribose) have been purified from mouse testis nuclei. Nuclear proteins were labelled in vitro with [14C]ribose and [3H]adenine, extracted with 5% (v/v) HClO4 and 0.25 M-HCl and separated by ion-exchange chromatography. Non-histone proteins were found to be the major acceptors in both the 5% (w/v)-HClO4-soluble and 5%-HClO4-insoluble HCl-extractable fractions. Of the two groups of non-histone proteins associated with chromatin, the LMG (low-mobility-group) proteins were preferentially ADP-ribosylated. HMG (high-mobility group) proteins were labelled to lower specific radioactivity. Six LMG proteins were purified to approx. 90% homogeneity and were identified from their mobility on polyacrylamide gels at pH 2.9 and from their amino acid composition. The average length of the poly(ADP-ribose) chain was estimated to be four to six repeating ADP-ribose units. It is suggested that ADP-ribosylation of LMG proteins, a long-neglected group of chromatin-associated proteins, is important during spermatogenesis for the production of spermatozoa with intact and competent DNA.

False-negative results for urinary phenothiazines and imipramine in Forrest's qualitative assays.

1980 ◽  
Vol 26 (2) ◽  
pp. 345-347 ◽  
Author(s):  
G P James ◽  
M H DJang ◽  
H H Hamilton
Keyword(s):  
Ascorbic Acid ◽  
Ion Exchange ◽  
Exchange Resin ◽  
Anion Exchange Resin ◽  
False Negative ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Negative Results ◽  
False Negative Results

Abstract When a series of patients' urine samples supplemented in vitro with chlorpromazine or imipramine was assayed with the Forrest qualitative assays, we observed an occasional false-negative result, which we found was attributable to interference by ascorbic acid. It interferes with the reagent, not with the analytes, in both assays. We easily eliminated this interference with the phenothiazine test by using an anion-exchange resin. Eliminating the interference with the assay for imipramine, however, is more difficult; false-negative results can be obtained even after ion-exchange chromatography if the imipramine concentration is less than 50 mg/L.

Isolation of a spermatozoal immobilization factor from Staphylococcus aureus filtrates

2009 ◽  
Vol 55 (7) ◽  
pp. 874-878 ◽  
Author(s):  
Vijay Prabha ◽  
Tanushree Gupta ◽  
Siftjit Kaur ◽  
Navchetan Kaur ◽  
Sushila Kala ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Permeation Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Spermatozoa ◽  
Infertile Woman ◽  
Ammonium Sulfate Precipitation ◽  
Tail Region ◽  
Sperm Immobilization

Staphylococcus aureus isolated from the cervix of an infertile woman was found to cause complete immobilization of human spermatozoa in vitro. Only the cell culture and cell-free supernatant showed immobilization activity, indicating that the sperm immobilization factor might be released extracellularly by the organism because no activity was observed with the washed cells. Heat treatment of the supernatant at 60 °C for 10 min waived its immobilizing activity, indicating that the active component may be a protein. The bioactive molecule from the supernatant was purified to homogeneity by ammonium sulfate precipitation, gel permeation chromatography, and ion exchange chromatography. Sperm immobilization factor (SIF) was found to be an ~20 kDa protein. SIF at a concentration of 10 µg/mL was required to cause 100% immobilization of human spermatozoa after 30 min of incubation at 37 °C, whereas a concentration of 150 µg/mL caused immediate immobilization, and a concentration of 200 µg/mL resulted in instant loss of viability of human spermatozoa, observed by eosin–nigrosin staining. Scanning electron microscopy showed that the treatment of human spermatozoa with SIF caused multiple defects in the head, midpiece, neck, and tail region of human spermatozoa.

THE UPTAKE OF TESTOSTERONE AND ZINC IN VITRO BY THE HUMAN BENIGN HYPERTROPHIC PROSTATE

1973 ◽  
Vol 58 (3) ◽  
pp. 405-419 ◽  
Author(s):  
M. JOAN REED ◽  
S. R. STITCH
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Zinc Binding ◽  
Supernatant Fraction ◽  
Low Molecular Weight ◽  
Molecular Weight Peak ◽  
Steroid Binding

SUMMARY The uptake of 65Zn and [1,2-3H]testosterone by minced tissue of human benign hypertrophic prostates and the subcellular distribution of radioactivity were examined. The nature of steroid and 65Zn binding by the cytosol (105000 g supernatant) fraction was investigated by gel filtration, ion-exchange chromatography and electrophoresis. It was found that steroid binding after incubation at 4°C was specific. One or two regions of steroid binding were observed after gel filtration of the cytosol using Sephadex G-200, depending upon incubation conditions. Binding of 65Zn was found in the low molecular weight peak after G-200 gel filtration. Equimolar CdCl2 and 65ZnCl2 were incubated with [1,2-3H]testosterone and minced tissue and the cytosol was subjected to gel filtration. Compared with control values, without CdCl2, reduction of 65Zn binding by about 50% occurred, while binding of 3H-labelled steroid was unaffected. Electrophoresis and ion-exchange chromatography showed that 65Zn and 3H-labelled steroid were bound to different proteins. A sample of the zinc-binding protein was prepared by ion-exchange chromatography and the homogeneity was checked by electrophoresis.

scholarly journals Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1255-1262 ◽  
Author(s):  
W Shi ◽  
BH Chong ◽  
CN Chesterman
Keyword(s):  
Cross Reactivity ◽  
Anticardiolipin Antibodies ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glycoprotein I ◽  
Activated Platelets ◽  
Major Interest ◽  
Thrombin Activation ◽  
Beta 2

Abstract Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid- dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]- iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]- ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2- GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

scholarly journals Analysis of histones from the yeast Saccharomyces carlsbergensis

1979 ◽  
Vol 177 (3) ◽  
pp. 917-923 ◽  
Author(s):  
A Pastink ◽  
T A Berkhout ◽  
W H Mager ◽  
R J Planta
Keyword(s):  
Basic Protein ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Saccharomyces Carlsbergensis ◽  
Histone Proteins ◽  
Chromosomal Proteins ◽  
Complete Set ◽  
Higher Eukaryotes ◽  
Main Components ◽  
Eukaryotic Organisms

Basic chromosomal proteins were isolated from the chromatin of the yeast Saccharomyces carlsbergensis by extraction with H2SO4 and were purified by ion-exchange chromatography. Electrophoresis of the purified fraction on acetic acid/urea gels revealed the presence of four main components. These four proteins were identified as histones H2A, H2B, H3 and H4 on the basis of their amino acid composition, molecular weight and solubility properties, all of which are very similar to the corresponding properties of the various histone proteins from other eukaryotic organisms. A fifth basic protein could be isolated from yeast chromatin by extraction with HClO4. The available evidence indicates this protein to be an H1-type histone. Yeast thus appears to contain a complete set of histone proteins which are strongly homologous to the histones occurring in higher eukaryotes.

Intestinal Absorption of Dipeptides Containing Glycine, Phenylalanine, Proline, β-Alanine or Histidine in the Rat

1973 ◽  
Vol 45 (6) ◽  
pp. 849-858 ◽  
Author(s):  
D. J. Boullin ◽  
R. F. Crampton ◽  
Christine E. Heading ◽  
D. Pelling
Keyword(s):  
Intestinal Absorption ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Peptide Absorption ◽  
Physiological Conditions ◽  
Anaesthetized Rats ◽  
Tissue Homogenates ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

1. The intestinal absorption of carnosine, glycylglycine, glycyl-d-phenylalanine, glycyl-l-phenylalanine, glycyl-l-proline and l-prolylglycine were investigated after intraluminal injection of dipeptide into anaesthetized rats. 2. With all six dipeptides, the intact substance was detected by ion-exchange chromatography in blood samples taken from the superior mesenteric vein. 3. The rate of hydrolysis of the dipeptides in tissue homogenates was measured in vitro. 4. The relative rates of hydrolysis varied by a factor of 300; there was an apparent inverse relationship between rate of hydrolysis and detection of intact peptide. 5. Peptide absorption was accompanied by increases in venous concentrations of the component amino acids, which appeared in proportions appropriate to the view that peptide absorption preceded hydrolysis. 6. It is suggested that slowly hydrolysed dipeptides may pass intact through the intestine wall under physiological conditions.

scholarly journals Protein-bound phosphorylserine in acid hydrolysates of brain tissue. The determination of [32P]phosphorylserine by ion-exchange chromatography and electrophoresis

1971 ◽  
Vol 121 (4) ◽  
pp. 597-600 ◽  
Author(s):  
D. A. Jones ◽  
R. Rodnight
Keyword(s):  
Ion Exchange ◽  
Specific Radioactivity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Exchange Method ◽  
Unknown Composition ◽  
Ion Exchange Method ◽  
Guinea Pig Brain ◽  
Cortical Slices

1. Partial acid hydrolysates of proteins derived from cortical slices of guinea-pig brain were divided into two parts and fractionated by ion-exchange chromatography and high-voltage electrophoresis. 2. The apparent yield of protein-bound phosphorylserine by the ion-exchange method was about three times that obtained by electrophoresis. 3. The specific radioactivity of phosphorylserine isolated from 32P-labelled slices by electrophoresis was twice that isolated by chromatography. 4. The discrepancies were found to be due to the presence of unlabelled phosphates of unknown composition in the ‘phosphorylserine’ fraction obtained by the ion-exchange method. 5. Electrical stimulation of slices respiring in the presence of [32P]phosphate increased the specific radioactivity of the total phosphate in the chromatographic ‘phosphorylserine’ fraction by 53±11%, as compared with only 19±5% for the phosphorylserine isolated by electrophoresis.

Isolation of Fasciola hepatica haemoglobin

Parasitology ◽  
1995 ◽  
Vol 111 (2) ◽  
pp. 209-215 ◽  
Author(s):  
S. McGonigle ◽  
J. P. Dalton
Keyword(s):  
Fasciola Hepatica ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Haemoglobin Molecule

SUMMARYA haemoprotein released in vitro by adult Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose. The molecule, with an apparent molecular weight of > 200 kDa, contains a haem group and has absorption spectra characteristics similar to haemoglobins. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica haemoglobin and other vertebrate or invertebrate haemoglobins. Antibodies to the haemoglobin molecule can be detected in the sera of F. hepatica-infected bovines as early as 1 week after infection.

scholarly journals Isolation and characterization of pig enamelins

1987 ◽  
Vol 243 (2) ◽  
pp. 385-390 ◽  
Author(s):  
H Limeback
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Binding Studies ◽  
Isolation And Characterization ◽  
Sequential Extraction Procedures ◽  
Enamel Proteins

Enamel proteins were extracted from pig developing enamel by sequential extraction procedures. Two proteins identified as enamelins by slab-gel electrophoresis (Mr 67,000 and 63,000) were separated from amelogenins by gel sieving and ion-exchange chromatography. Their enamelin characteristic was confirmed by hydroxyapatite-binding studies and amino acid analysis. Degradation of extracted enamel proteins was also studied in vitro. The larger of the two enamelins appeared to be resistant to degradation by endogenous enamel proteinases. Hydroxyapatite showed strong binding with the enamelins, but did not prevent the degradation of the Mr-63,000 enamelin. These results indicate that at least one high-Mr enamelin in pig developing enamel is a source of enamelin breakdown products.

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