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Biomedical subjects

E C Heath

Publications and source records attributed to E C Heath.

At least 37 records · Page 2Linked to original sources
PubMed1979

A hybrid protein containing the toxic subunit of ricin and the cell-specific subunit of human chorionic gonadotropin. I. Synthesis and characterization.

In order to study the mechanisms involved in the interaction of cell-surface components and effector proteins, we synthesized a double-labeled dimeric hybrid molecule consisting of the toxic A subunit of the lectin ricin and the cell-specific beta subunit of human chorionic gonadotropin (hCGbeta). Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of hCGbeta and toxin subunit A was prepared in 30% yield relative to hCGbeta input. The dimeric protein, purified by gel filtration and ion exchange chromatography, was characterized by: (a) sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol, (b) analysis by gel filtration chromatography in the presence and absence of 2-mercaptoethanol, and (c) precipitation with antibodies specific for hCG and subunit A.

Animals↗
PubMed1979

A hybrid protein containing the toxic subunit of ricin and the cell-specific subunit of human chorionic gonadotropin. II. Biologic properties.

The biologic properties of a purified hybrid protein containing the toxic subunit A of ricin and the cell-specific subunit of human chorionic gonadotropin (hCGbeta) in a disulfide conjugate is reported. The biologic activity of the hybrid has been studied by assaying inhibition of protein synthesis in rat R2C Leydig cells and mouse L-cells. The results indicate that for the A subunit to exhibit its toxic effect it must first bind to cells through receptors which are specific for the beta subunit of the hybrid. Thus, only cells with beta-type receptors are sensitive to the toxic effects, which hCG is capable of blocking, while cells which are not target cells for hCG are not subject to the toxic effects. Thus it is concluded that the disulfide conjugate may act as a functional analog of ricin toxin but with altered cell specificity. Or, alternately, the hybrid may be regarded as an analog of hCG but with drastically altered physiologic effects.

Animals↗
PubMed1979

De novo biosynthesis of an enzymatically active precursor form of bovine pancreatic RNase.

The de novo biosynthesis of RNase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) was studied in a cell-free rabbit reticulocyte translation system using a polyadenylylated fraction of mRNA isolated from bovine pancreas. Analysis of the [35S]methionine-labeled translation products of pancreas mRNA by polyacrylamide gel electrophoresis indicated the presence of several proteins, one of which corresponded to 16,500 daltons, or approximately 2800 daltons greater than native RNase A. This protein was specifically precipitated from the mixture of products by anti-RNase antibodies. Partial sequence determination of the NH2-terminal region of the anti-RNase antibody-precipitable species indicated that it is a precursor form of RNase A with 25 additional amino acids on its NH2 terminus. The precursor nature of the protein was confirmed by demonstration that a mixture of RNase A and a glycosylated form of the enzyme, RNase B, is formed when translation of the mRNA is conducted in the presence of dog pancreas membranes. Assay of the putative precursor form of RNase for catalytic activity with polycytidylic acid as substrate indicated that the protein has a specific enzymatic activity identical to that of native RNase A.

Animals↗
PubMed1978

A selective and sensitive assay for urinary metanephrine and normetanephrine using gas chromatography mass spectrometry with selected ion monitoring.

Vapor phase methodology for quantitative analysis of urine for metanephrine and normetanephrine as the pentafluoropropionyl derivative is described. The use of selected ion monitoring provided sufficient sensitivity and selectivity that milliliter aliquots of urine required only a simple scheme of sample processing; preliminary purification consisted of percolation through a column of XAD-2 followed by solvent extraction. The sample extracts were converted to the pentafluoropropionyl derivatives and analyzed by gas chromatography mass spectrometry with selected ion monitoring. Quantitative analysis was based on alpha[2H2]-beta-[2H1]metanephrine and alpha-[2H2]-beta-[2H1]normetanephrine which were added to the urine sample at the outset. The calibration plots for metanephrine and normetanephrine were linear from 10 ng ml-1 to 2000 ng ml-1 of urine permitting the detection of metanephrine and normetanephrine levels throughout the normal range and well into the subnormal range. Examples of application of the methodology to clinical investigation are described. The overall reproducibility of the sample processing methodology and instrumentation was assessed by replicate analyses of the same urine sample over a period of several weeks; the coefficient of variation was +/- 2.5% (n = 8).

Chromatography, Gas↗
PubMed1977

Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations.

Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.

Animals↗
PubMed1977

Glycoprotein biosynthesis in myeloma cells. Characterization on nonglycosylated immunoglobulin light chain secreted in presence of 2-deoxy-D-glucose.

The biosynthesis and secretion of a glycosylated, K-type immunoglobulin light chain (K-46) was studied in a mouse myeloma tumor, mineral oil plasmacytoma-46B. Viable single cell suspensions were prepared from excised tumors and optimal conditions were established for incorporation of amino acid and carbohydrate precursors into the protein synthesized and secreted by the cells. The glucose analog, 2-deoxy-D-glucose, was utilized as an inhibitor of glycosylation to determine the role of glycosylation in the biosynthesis, intracellular transport, and export of the protein from the cell. It was determined that 6 mM 2-deoxyglucose prevents the incorporation of glucosamine, mannose, and galactose into secreted protein, but permits the incorporation of leucine at approximately 40% of control values. The nonglycosylated protein, secreted in the presence of 2-deoxyglucose, was characterized as a nonglycosylated form of K-46 light chain by the following criteria: (a) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, (b) reactivity of the nonglycosylated protein with antisera prepared against native, fully glycosylated, K-46 light chain, (c) analysis of the protein by gel filtration techniques, (d) behavior of the protein on lectin-derivatized Sepharose, and (e) analysis of tryptic peptides derived from the protein. We have concluded that 2-deoxyglucose-inhibited cells synthesize and secrete the normal polypeptide chain of K-46 devoid of its carbohydrate side chain indicating that glycosylation is not an essential step in the biosynthesis, intracellular transport, or export of this protein that is normally synthesized and secreted in a glycosylated form. Under conditions of 2-deoxyglucose inhibition, the nonglycosylated form of K-46 light chain constitutes a significantly greater proportion of accumulated intracellular protein, suggesting that the biosynthesis of the polypeptide chain of K-46 light chain proceeds at a nearly normal rate, but that the absence of the carbohydrate side chain of the protein retards, but does not prevent, the intracellular transport of the protein and its export from the tumor cell.

Animals↗
PubMed1975

Isolation and characterization of a bacteriophage polysaccharide.

A high molecular weight heteropolysaccharide, composed of glucose, glucuronic acid, N-acetylglucosamine, and mannose in an approximate molar ratio of 1:2:2:5, respectively, was isolated from phage K-2 and from the soluble fraction of phage-infected Aerobacter aerogenes lysates. Treatment of pure phage with 8 M urea at 4 degrees quantitatively solubilizes the bound polysaccharide and capsular polysaccharide (Yurewicz, E.C., Ghalambor, M.A., Duckworth, D.H., and Heath, E.C. (1971) J. Biol. Chem. 246, 5607-5616) with the release of only traces of other phage constituents; on this basis, it was concluded that the polysaccharide, like the the glycanohydrolase, is externally localized in the phage structure. Phage polysaccharide and glycanohydrolase fractionate similarly on ion exchange resins and gel electrophoresis in sodium dodecyl sulfate, but each may be purified to homogeneity by the procedures employed. The biosynthesis of the polysaccharide was shown to be uniquely dependent upon phage K-2 infection by: (a) absence of the polysaccharide in cells, the culture filtrate, or sonicated extracts of uninfected cells; (b) kinetics of polysaccharide synthesis following phage infection; and (c) isotopic double-labeling experiments that demonstrated the synthesis of polysaccharide only after initiation of phage replication in infected cells.

Acetylglucosamine↗
PubMed1974

The role of a dolichol-oligosaccharide as an intermediate in glycoprotein biosynthesis.

Incubation of mouse myeloma microsomes with GDP-[(14)C]mannose results in the biosynthesis of [(14)C]mannose phosphoryl dolichol [Baynes, J. W., Hsu, A.-F. & Heath, E. C. (1973) J. Biol. Chem. 248, 5693-5704] and a [(14)C]mannose- and N-acetylglucosamine (GlcNAc)-containing oligosaccharide derivative of dolichol. Thus, [(14)C]mannose phosphoryl dolichol and [(14)C]mannose-labeled oligosaccharide pyrophosphoryl dolichol were isolated from incubation mixtures by solubilization in 2% (w/v) Triton X-100 and the lipids were separated from small molecules by gel filtration fractionation. After removal of radioactive protein from the preparation, the two lipid derivatives were separated quantitatively by fractionation on a concanavalin A-Sepharose column; [(14)C]mannose phosphoryl dolichol was not retained by the affinity resin but [(14)C]mannose-oligosaccharide pyrophosphoryl dolichol adsorbed to the gel and was eluted with alpha-methylmannoside.[(14)C]Mannose-oligosaccharide pyrophosphoryl dolichol appeared to be homogeneous when fractionated on DEAE-cellulose and in several thin-layer chromatographic systems. Treatment of [(14)C]mannose oligosaccharide pyrophosphoryl dolichol with 10% (w/v) NH(4)OH at 100 degrees for 1 hr resulted in the formation of a water-soluble radioactive oligosaccharide phosphate which was isolated and characterized as [Man](5) --> [GlcNAc --> GlcNAc --> P. Incubation of [(14)C]mannose-oligosaccharide pyrophosphoryl dolichol with myeloma microsomal preparations results in the transfer, presumably, of the entire oligosaccharide to endogenous protein. Kinetic studies indicate that the dolichol derivatives serve as intermediates in the glycosylation of protein as follows: [Formula: see text]

Animals↗