The Development of a Radioimmunoassay for the Measurement of Urinary Tammhorsfall Glycoprotein in the Presence of Sodium Dodecyl Sulphate

1973 ◽  
Vol 44 (2) ◽  
pp. 163-179 ◽  
Author(s):  
Anne M. S. Grant ◽  
A. Neuberger
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Solid Phase ◽  
Excretion Rate ◽  
Sodium Dodecyl ◽  
Antibody Concentration ◽  
Low Concentrations ◽  
Freeze Dried ◽  
Ionic Detergent ◽  
Normal Humans

1. A specific and quantitative radioimmunoassay was developed for the measurement of low concentrations of human and rabbit Tamm—Horsfall glycoprotein in the presence of other proteins. Antibody-coated tubes were used as a solid phase in the assay and the optimum antibody concentration and duration of antibody coating were established. 2. Pure Tamm—Horsfall glycoprotein was labelled with 125I and, because of its apparent susceptibility to radiation damage, was labelled at weekly intervals. 3. Sodium dodecyl sulphate, an ionic detergent, was included in the assay at a final concentration of 0.0005% to disaggregate the glycoprotein. An overnight preincubation step in the presence of the detergent was necessary before the disaggregated glycoprotein solutions were allowed to react with the antibody. Pretreatment of the tracer with detergent was not necessary. 4. Two glycoprotein standards were prepared fresh for each assay from freeze-dried material. The average linear range of the assay was between approx. 150 ng/ml and 2.5 μg/ml. Albumin was only shown to interfere with the assay at concentrations greater than 100 μg/ml. 5. Urines were dialysed against water for 3 days before assay to remove inhibitory material. Urines were never frozen as this was found to affect the assay. 6. A recovery experiment showed that the pure freeze-dried standard behaved in an immunologically identical way to the urinary glycoprotein. 7. Human Tamm-Horsfall glycoprotein cross-reacted with guinea-pig anti-(rabbit Tamm—Horsfall) antiserum and rabbit Tamm—Horsfall glycoprotein cross-reacted with guinea-pig anti-(human Tamm—Horsfall) antiserum, but not with rabbit anti-(human Tamm—Horsfall) antiserum. This showed a partial immunological identity between Tamm-Horsfall glycoprotein from humans and rabbits which was only evident when the antiserum was raised in a third species. 8. The excretion rate of Tamm—Horsfall glycoprotein in normal humans was found to be 48.1 ± 9.6 (SD) mg/24 h for males and 50.5 ± 14.8 (SD) mg/24 h for females. The mean excretion rate of the glycoprotein in New Zealand White rabbits was 34.8 ± 7.9 mg/24 h.

scholarly journals Initiation and processing in vitro of the primary translation products of guinea-pig caseins

1979 ◽  
Vol 184 (2) ◽  
pp. 261-267 ◽  
Author(s):  
R K Craig ◽  
P A J Perera ◽  
A Mellor ◽  
A E Smith
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Secretory Proteins ◽  
Post Translational Modifications ◽  
Microsomal Membranes

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal ‘signal’-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

scholarly journals The identification and characterization of two separate carboxylesterases in guinea-pig serum

1983 ◽  
Vol 215 (1) ◽  
pp. 91-99 ◽  
Author(s):  
K Cain ◽  
E Reiner ◽  
D G Williams
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Enzyme Preparation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Selective Inhibitor ◽  
Nitrophenyl Phosphate ◽  
Phosphate Treatment ◽  
Pig Serum

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.

scholarly journals Phosphoenolpyruvate carboxykinase from guinea-pig liver mitochondria. Immunological evidence for increase in enzyme amount during neonatal development

1984 ◽  
Vol 221 (1) ◽  
pp. 105-111 ◽  
Author(s):  
S Wicheanvonagoon ◽  
I J Arinze
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Liver Mitochondria ◽  
Polyacrylamide Gels ◽  
Neonatal Development ◽  
Pig Liver ◽  
Guinea Pig Liver

Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.

scholarly journals Wheat-germ aspartate transcarbamoylase. The effect of ligands on the inactivation of the enzyme by trypsin and denaturing agents

1973 ◽  
Vol 131 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Robert J. Yon
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Low Concentrations ◽  
Alkaline Conditions ◽  
High Concentrations ◽  
Very High ◽  
Resistant State

In the absence of added ligands aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ is inactivated fairly rapidly by trypsin, by heat (60°C), by highly alkaline conditions (pH11.3) and by sodium dodecyl sulphate. Addition of UMP alone, at low concentrations, decreases the rate of inactivation by each of these agents significantly. Carbamoyl phosphate alone does not alter the rate of inactivation by trypsin and by the detergent, but it antagonizes the effect of UMP in protecting the enzyme against these agents. These results have been interpreted to mean that two conformational states are reversibly accessible to the enzyme, namely an easily inactivated state favoured in the presence of carbamoyl phosphate and a more resistant state favoured in the presence of UMP. In the absence of ligands the enzyme is in the easily inactivated conformation. At very high concentrations l-aspartate also protects the enzyme but to a smaller extent than UMP. Some implications of these results are discussed.

scholarly journals Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate

1982 ◽  
Vol 202 (2) ◽  
pp. 317-323 ◽  
Author(s):  
I H Mather ◽  
C H Sullivan ◽  
P J Madara
Keyword(s):  
Xanthine Oxidase ◽  
Sodium Dodecyl Sulphate ◽  
Milk Fat ◽  
Solid Phase ◽  
Degradation Products ◽  
Sodium Dodecyl ◽  
Skim Milk ◽  
Soluble Form ◽  
Mammary Tissue ◽  
Polyacrylamide Gels

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either ‘mitochondrial’ or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.

scholarly journals A phospholipid-deacylating system of bacteria active in a frozen medium

1976 ◽  
Vol 153 (1) ◽  
pp. 49-53 ◽  
Author(s):  
G P Hazlewood ◽  
R M C Dawson
Keyword(s):  
Oleic Acid ◽  
Low Temperature ◽  
Sodium Dodecyl Sulphate ◽  
Low Temperatures ◽  
Solid Phase ◽  
Sodium Dodecyl ◽  
Hydrolysis Rate ◽  
Bacterial Cells ◽  
Phospholipase Activity ◽  
Absolute Requirement

A phosphatidylcholine-deacylating system present in a Butyrivibrio species (probably fibrisolvens) shows appreciable activity at low temperatures with a maximum hydrolysis rate at—10 degrees C. 2. The rate at—10 degrees C is higher than at 39 degrees C unless the system at the latter temperature is stimulated by adding oleic acid or sodium dodecyl sulphate. 3. The low-temperature phospholipase activity has an absolute requirement for thiol reagents, e.g. cysteine, dithiothreitol or mercaptoethanol. 4. Ca2+, Mg2+ and Mn2+ stimulate the activity up to 10 mM, but EDTA inhibits; higher concentrations of Ca2+ also inhibit. 5. The enhancement of activity at low temperatures appears not to be associated with a crystalline change in the hydrated phospholipid substrate, but depends on the formation of a solid phase in the incubation medium which brings the substrate and bacterial cells into juxtaposition or causes fusion.

scholarly journals Studies of the denaturation and partial renaturation of ovalbumin

1972 ◽  
Vol 129 (3) ◽  
pp. 665-676 ◽  
Author(s):  
J. C. Holt ◽  
J. M. Creeth
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Optical Rotatory Dispersion ◽  
Rotatory Dispersion ◽  
Physical Parameters ◽  
Optical Rotatory ◽  
Guanidinium Chloride ◽  
Dispersion Parameters ◽  
Low Concentrations

1. The denaturation of ovalbumin by the reagents sodium dodecyl sulphate and guanidinium chloride was investigated, by following the changes in sedimentation velocity, optical rotatory dispersion and viscosity as a function of denaturant concentration. 2. With sodium dodecyl sulphate both the optical-rotatory-dispersion parameters a0 and b0 become more negative, the sedimentation coefficient decreases and the viscosity increases; significant differences in the denaturation profiles are observed. The change in each parameter is indicative of only limited denaturation. 3. With guanidinium chloride the transition occurs over the concentration range 1–4m: more extensive changes occur in all the physical parameters than with sodium dodecyl sulphate. The values of a0 and b0 are indicative of complete denaturation. Reduction by mercaptoethanol produces only minor further changes. 4. Renaturation was attempted from both denaturants, the removal of reagent being accomplished reversibly by controlled slow dialysis. Partial renaturation was observed, but aggregated or insoluble material was produced in both cases at relatively low concentrations of denaturant. Similar behaviour was observed with fully reduced protein in guanidinium chloride–mercaptoethanol; complete renaturation could not be brought about even at very low protein concentrations.

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