PubMed Health⌕ Search

Biomedical subjects

W J Gullick

Publications and source records attributed to W J Gullick.

155 records · Page 9Linked to original sources
PubMed1983

Comparison of the subunit structure of acetylcholine receptors from muscle and electric organ of Electrophorus electricus.

The acetylcholine receptors of the electric organ and muscle tissues of Electrophorus electricus are composed of alpha, beta, gamma, and delta subunits. Receptor subunits from the two tissues were compared by peptide mapping with monoclonal antibodies, an affinity-labeling reagent, and a lectin to characterize particular peptide fragments. These experiments indicate that the corresponding receptor subunits from the two tissues are extensively homologous or identical throughout their amino acid sequences. Small differences in the electrophoresis of peptide fragments of alpha subunits between the two tissues occurred on fragments which bound labeled lectin. These results suggest that the acetylcholine receptors in electric organ and muscle tissues of Electrophorus differ in structure only by minor posttranslational modifications perhaps involving carbohydrate.

Animals↗
PubMed1982

Structural similarities between acetylcholine receptors from fish electric organs and mammalian muscle.

Acetylcholine receptors from fish electric organ tissue and mammalian muscle were compared by peptide mapping. The alpha subunits from receptors of Torpedo californica and Electrophorus electricus electric organ tissue were digested with V8 protease and the resulting fragments separated on polyacrylamide gels and stained for protein or for carbohydrate. 125I-Labeled alpha subunits of acetylcholine receptors from Electrophorus electric organ tissue and bovine muscle were digested with V8 protease, and the resulting fragments were also separated on polyacrylamide gels. Intact receptors from both the fish electric organs and mammalian muscle were labeled with [4-(N-maleimido) benzyl]tri[3H]-methylammonium iodide which binds specifically to the acetylcholine binding site on alpha subunits, and the isolated alpha subunits were subjected to the same peptide mapping procedure. The fragment patterns produced were stained for protein and fluorographed to identify active site containing polypeptides. None of these peptide mapping approaches revealed extensive homologies between alpha subunits. Intact and V8 proteolyzed sodium dodecyl sulfate denatured receptors from Torpedo and Electrophorus electric organs and bovine muscle were electrophoretically transferred to diazophenyl thioether paper and probed with antisera to Torpedo receptor subunits and two monoclonal antibodies. Unique fragment patterns were obtained with each antisubunit serum. A fragment of the same size was derived from the beta subunit of each acetylcholine receptor and was shown to specifically bind the same monoclonal antibody in each case. These results indicate that only in the beta subunits from all of the species examined is a large sequence nearly identical. However, it is likely that corresponding receptor subunits from receptors of all of these species have extensive structural homologies.

Animals↗
PubMed1982

The antigenic structure of the acetylcholine receptor from Torpedo californica.

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.

Animals↗
PubMed1981

Fragmentation of a mollusc haemocyanin with plasmin and immunological identification of the fragments.

Haemocyanin from the gastropod mollusc Lymnaea stagnalis (pond snail) was partially digested with plasmin under a variety of experimental conditions and the products of digestion analysed by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Fragments were obtained corresponding to one, two, three, four and five oxygen-binding domains, one domain having a mol.wt. of approx. 50000 and containing 2 ions of Cu. The fragments obtained after extensive digestion were non-identical immunologically, and summation of their molecular weights allowed a minimal mol.wt. of 413000 to be calculated for the original, undigested, eight-domain polypeptide chain. The use of mild-digestion conditions allowed the time course and sequence of the digestion to be monitored. An initial cleavage gave a three-domain and a five-domain fragment. The three-domain fragment was resistant to further digestion. The five-domain fragment could be digested further to give, successively a four-domain, a three-domain, and finally a two-domain fragment, single-domain units being cleaved. These data form the basis for a proposed sequence for the different domains in the original chain.

Animals↗
PubMed1981

Monoclonal antibodies as probes of acetylcholine receptor structure. 1. Peptide mapping.

The isolated subunits of the acetylocholine receptor from Torpedo californica were digested with proteolytic enzymes, and the resulting polypeptide fragments were analyzed by gel electrophoresis. We have identified those fragments which contain carbohydrate and those from the alpha subunit which are labelled with the acetylcholine binding site specific reagent [4-(N-maleimido)benzyl]tri[3H]methylammonium iodide. We have tested several monoclonal antibodies raised to the acetylcholine receptor from torpedo, some of which react with the denatured subunits [Tzartos, S.J., & Lindstrom, J.M. (1980) Proc. Natl. Acad. Sci. U.S.A.77, 755; Tzartos, S.J., & Lindstrom, J.M. (1981) in Monoclonal antibodies in Endocrine Research (Fellows, R., & Eisenbarth, G., Eds.) Raven Press (in press)]. The binding specificities of these antibodies to radioiodinated proteolytically generated fragments of the alpha subunit were determined by immunoprecipitation followed by gel electrophoresis. The antibodies tested fell into at least three main groups on the basis of their binding specificities. These antibodies were also tested for their capacity to bind to acetylcholine receptor solubilized in Triton X-100, sodium cholate, or sodium cholate supplemented with exogenous lipids. A monoclonal antibody raised to the denatured delta subunit, was tested for its ability to select radioiodinated proteolytic fragments of these subunits. These molecules provide probes for many sites on the acetylcholine receptor with affinities and specificities comparable to alpha-neurotoxins.

Animals↗
PubMed1979

Characterization of domains obtained from a mollusc haemocyanin by limited proteolytic digestion.

The haemocyanin from the freshwater gastropod Lymnaea stagnalis was digested with proteolytic enzymes under conditions where it existed as whole (native) molecules (mol.wt. approx. 9 X 10(6)), or as one-tenth molecules. Digestion of whole molecules yielded a fragment of mol.wt. approx. 110,000 believed to correspond to the 'collar' of the molecule, and an aggregate some 20--30 times the size of the original native molecule formed by end-to-end polymerization of the molecule after removal of the collar. Digestion of one-tenth molecules yielded a mixture of products that could be separated into three fractions by gel filtration. Analysis of these by sodium dodecylsulphate/polyacrylamide-gel electrophoresis revealed that they typically contained two or three components. The collar fragment was present as a component of the intermediate-molecular-weight fraction, and it dissociated on sodium dodecyl sulphate/polyacrylamide gels to give two bands corresponding to apparent mol.wts. 65,000 and 60,000. The c.d. spectra of the separated fractions were recorded and fitted with Gaussian curves by a computer procedure. The fractions each possessed distinct c.d. spectra, by which they could be identified: the collar-fragment c.d. and absorption spectra showed the most striking differences compared with those of the other fragments. The results were interpreted in terms of the postulated existence, within the haemocyanin molecule, of multi-domain structures, each comprising a single polypeptide chain of mol.wt. 200,000--300,000.

Animals↗
PubMed1979

Planorbis corneus haemoglobin. Circular dichroism and susceptibility to proteases.

The purified haemoglobin of Planorbis corneus was subjected to protease digestion and the resulting products characterised by gel filtration and detergent-gel electrophoresis. Small functional subunits of molecular weights approximately 20,000 were obtained corresponding to a single haem group, but multiples of this unit were also always obtained even at high proteolytic enzyme: haemoglobin ratios. This suggested that the subunits of the native molecule (one-tenth containing perhaps ten O2-binding sites) were made up of single-binding site domains linked by regions of polypeptide chains having different susceptibilities to proteases. The far ultraviolet CD of the native haemoglobin indicated the presence of a high helix content (75--80%) in the protein. The near ultraviolet and visible CD spectra of oxy- deoxy-, and CO-haemoglobin were reported. Planorbis haemoglobin CD was more like that of vertebrate haemoglobins than that of haemoblobins. Nevertheless the Soret CD of Planorbis oxyhaemoglobin had only about half the rotational strength of that of human haemoglobin A, and was halved again upon removal of the ligand. Also in contrast to Lumbricus and human haemoglobins there was only a small decrease in rotational strength in the 260 nm band when Planorbis oxyhaemoglobin was deoxygenated.

Animals↗
PubMedDate not supplied

Expression of HER-2/neu oncoprotein in human breast cancer: a comparison of immunohistochemical and western blot techniques.

Three hundred and one primary breast cancers from patients with tumor infiltrated lymph nodes were analyzed for the presence of HER-2/neu oncoprotein by two procedures: Western blot (WB) and immunohistochemistry (IHC). Overexpression of this protein was found by WB in 16.6% of the tumors, and by IHC in 16.3%. Concordance between the two methods was found in 95% of tumors (286/301). In 7 cases we found HER-2/neu by IHC but not by WB, while the opposite was found in the remaining 8 patients. This discrepancy was found mainly in samples with HER-2/neu values just above the cut points and were therefore close to the sensitivity limits of the procedures used here. This study helps to define the parameters that should be considered to evaluate the immunostaining for HER-2/neu as positive (i.e., membrane staining, IHC score of 2 or more). The results obtained by both techniques were correlated with several currently used prognostic factors. Higher HER-2/neu protein expression was found in tumors lacking estrogen or progesterone receptors, in tumors with high S-phase fraction and in patients with more than 3 positive lymph nodes. In contrast, no relationship was found between overexpression of this protein and tumor size, ploidy, or age of the patient. Patients with elevated HER-2/neu expression showed a significantly worse overall survival by both methods, IHC (p = 0.05) and WB (p = 0.001). In conclusion, there is very high agreement between IHC and WB when measuring expression of HER-2/neu and both techniques showed prognostic significance.

Biomarkers, Tumor↗