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Biomedical subjects

E C Heath

Publications and source records attributed to E C Heath.

At least 19 recordsLinked to original sources
PubMed1988

Relative and absolute bioavailability of prednisone and prednisolone after separate oral and intravenous doses.

A randomized, four-way cross-over study was conducted in eight healthy male volunteers to determine the relative and absolute bioavailability of prednisone (PN) and prednisolone (PL). PN and PL were administered as single, oral 10-mg tablet doses and as 10-mg zero-order 0.5-hour intravenous infusions. Comparable mean PN and PL maximum plasma concentrations (Cmax), times for Cmax, areas under the plasma concentration-time curves (AUC), and apparent elimination rate constants between tablet treatments demonstrated that PN and PL tablets were bioequivalent. Absolute bioavailability (F) determinations based on plasma PL concentrations were independent of which IV treatment was used as reference and indicated complete systemic availability of PL from both PN and PL tablets. However, F based on plasma PN data was contradictory. Using IV PN as reference, approximately 70% systemic availability was observed from both tablets, whereas using IV PL as reference, systemic availability was greater than unity. PN and PL are model compounds that exemplify the difficulties involved in accurately determining the relative and absolute bioavailability of substances that undergo reversible metabolism.

Administration, Oral↗
PubMed1986

Bovine angiotensin-converting enzyme: amino-terminal sequence analysis and preliminary characterization of a hybridization-selected primary translation product.

Bovine lung angiotensin-converting enzyme was isolated in pure form and the sequence of the first twenty-two NH2-terminal amino acids determined. Oligonucleotides, complementary to a selected portion of the NH2-terminal amino acid sequence of the bovine glycoprotein (Mr 145,000), were synthesized and used for hybridization selection of angiotensin-converting enzyme mRNA. The hybridization-selected mRNA programmed the in vitro synthesis of a single polypeptide (Mr 130,000) that was specifically immunoadsorbed by anti-bovine enzyme antibodies. Preliminary sequence analysis of the primary translation product suggests that bovine angiotensin-converting enzyme is synthesized without a transient NH2-terminal signal sequence.

Amino Acid Sequence↗
PubMed1986

Evidence for posttranslational O-glycosylation of fetuin.

Fetuin, a major glycoprotein in the serum of fetal calves that contains three N-linked and three O-linked carbohydrate side chains, was found to be synthesized in the liver with an 18 amino acid signal peptide, Met-X-X-X-X-Leu-Leu-X-Cys-Leu-Ala-X-Leu-X-X-Cys-X-X, and to undergo cotranslational N-glycosylation. In order to examine O-glycosylation, fetuin peptidyl-tRNA was purified from liver and analyzed for O-linked carbohydrate by quantitating the released [3H]GalNAcitol produced after beta-elimination in the presence of NaB3H4. Within the limits of the assay, less than 1.3% of the O-linked chains had been initiated. Additionally, rough microsomes were used to program a cell-free protein synthesis system. A radiolabeled fetuin intermediate was isolated by immunoprecipitation and shown to contain N-linked carbohydrate by binding to concanavalin A and by susceptibility to cleavage by endoglycosidase H. However, this fetuin intermediate was not detectably bound (less than 1%) by GalNAc-specific lectins, which were shown to bind asialoagalactofetuin. These results suggest that O-glycosylation of fetuin is a posttranslational event.

Amino Acid Sequence↗
PubMed1986

Biosynthesis, processing, and secretion of M and Z variant human alpha 1-antitrypsin.

The Z genetic variant of human alpha 1-antitrypsin (alpha 1AT) is associated with decreased serum alpha 1AT levels, hepatic inclusion bodies, and an increased risk of lung and liver disease. We studied the biosynthesis, processing, and secretion of normal and Z variant alpha 1AT in cell-free translation systems, reconstituted in vitro processing systems, and in the Xenopus oocyte secretory system. Human liver mRNA was prepared from normal subjects (PiMM) and from individuals homozygous for alpha 1AT deficiency (PiZZ). Cell-free translation resulted in the synthesis of 49,000-Da preproteins with a 23-amino acid signal sequence. The genetic variants were synthesized at comparable levels and could be distinguished on the basis of charge. The majority of the amino acids in the ZZ signal peptide were identified and found to be the same as those comprising the MM signal sequence. These proteins were co-translationally processed with similar efficiency by dog pancreas microsomes, producing 52,000-Da glycoproteins which were completely translocated across the endoplasmic reticulum membrane. When the human liver RNA preparations were injected into Xenopus oocytes, both of the alpha 1AT variants were synthesized intracellularly and alpha 1AT was detected in the medium of all oocytes injected with MM RNA. However, the Z variant accumulated within the microsomal vesicles of the cell and was undetectable or present at decreased levels in the medium. We conclude that the single amino acid substitution in the Z variant of alpha 1AT does not affect its synthesis or co-translational processing but that it strongly affects its transport from the rough endoplasmic reticulum through the secretory pathway.

Amino Acids↗
PubMed1986

Structural features of bovine fetuin revealed from analysis of the primary translation product: anomalous behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is due largely to peptide and not solely to carbohydrate.

Native bovine fetuin is a major alpha 1-glycoprotein in the serum and cerebrospinal fluid of fetal calves. We previously reported (Johnson, W.V., and Heath, E.C. (1986) Biochemistry 25, 5518-5525) the purification of the primary translation product for fetuin (prefetuin) from a rabbit reticulocyte cell-free translation system and showed that prefetuin contains an 18 amino acid signal peptide. Here we report that although the apparent sodium dodecyl sulfate (SDS) gel molecular weights of fetuin and prefetuin are 64,000 and 49,000, respectively, when analyzed by gel filtration chromatography under denaturing and reducing conditions, molecular weight values of 48,000 and 40,000 were found for native fetuin and the nonglycosylated prefetuin, respectively. These molecular weight values are in agreement with those expected on the basis of the sedimentation diffusion data of Spiro (Spiro, R.G. (1960) J. Biol. Chem. 235, 2860-2869) and indicate that the polypeptide moiety makes a major contribution toward the anomalous SDS gel electrophoretic mobility of fetuin. Therefore, the carbohydrate moiety is not solely responsible for this property. Edman degradation of [35S]methionine-labeled prefetuin indicated that the N-terminal residue is the only methionine present in prefetuin; native fetuin lacks methionine. Additionally, hydroxylamine cleavage of an Asn-Gly bond in prefetuin localized one of the N-linked carbohydrate side chains to the middle of the polypeptide chain of native fetuin.

Animals↗
PubMed1986

Biosynthesis and processing of a variant surface glycoprotein from Trypanosoma brucei brucei.

Iowa trypanosome antigen type (IaTat) 1.2 variant surface glycoprotein (VSG) is synthesized in vitro as a Mr 54,000 preprotein that contains a 31-amino-acid signal peptide. Translation of mRNA in the presence of either dog pancreas or trypanosome microsomal membranes results in cotranslational cleavage of the signal peptide and addition of core oligosaccharide side chains to the protein. Analysis of these products on sodium dodecyl sulfate (SDS)-gels indicates that removal of the signal peptide (Mr 3200) is almost exactly compensated for by an increase in molecular weight due to carbohydrate addition. Pulse-chase experiments in cultures of isolated trypanosomes indicate that two IaTat 1.2 VSG species (Mr 58,000 and 60,000) occur in vivo. When glycosylation is inhibited by incubation of trypanosomes with tunicamycin, a single Mr 50,000 polypeptide is immunoprecipitated. The multiple protein species, therefore, arise from heterogeneity in carbohydrate side chains whose synthesis and transfer to the protein are tunicamycin sensitive. Sequence analysis verified that both species of VSG contain identical amino-terminal sequences. Further post-translational processing of IaTat 1.2 VSG includes addition of phosphate and myristic acid residues, both of which have been shown to be located in the immunologically cross-reactive determinant at the carboxyl terminus of the protein. Exposure of this attachment site requires post-translational proteolytic removal of a 17-amino-acid peptide from the carboxyl terminus of an intermediate form of VSG.

Amino Acid Sequence↗
PubMed1985

The relationship between rat major acute phase protein and the kininogens.

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.

Acute-Phase Proteins↗
PubMed1985

A novel proteolytic activity in serum processes rat prohaptoglobin.

The heterotetrameric plasma glycoprotein rat haptoglobin previously was shown to be synthesized by hepatocytes in a precursor form, prohaptoglobin, which contains one alpha-subunit region and one beta-subunit region. Two of these molecules, each with a molecular weight of 45,000, are joined by a disulfide bond and subsequently the subunit regions of each polypeptide are separated by site-specific proteolysis, yielding the tetrameric native protein. Although some of this processing occurs intracellularly, a substantial proportion of the prohaptoglobin is secreted [J. M. Hanley, T. H. Haugen, and E. C. Heath (1983) J. Biol. Chem. 258, 7858-7869]. However, a proteolytic activity was found in rat plasma and serum which also is capable of site-specific cleavage of prohaptoglobin. Further investigation of this novel activity has demonstrated that it cleaves prohaptoglobin accurately, in the same site-specific manner as the intracellular protease, and that it most likely is not a serine protease or a metalloenzyme but can be inhibited by sulfhydryl-reactive compounds. Furthermore, it appears to be synthesized and secreted by hepatocytes, and thus may be identical to the intracellular processing protease.

Alkaloids↗
PubMed1984

Nucleotide sequence of rat haptoglobin cDNA. Characterization of the alpha beta-subunit junction region of prohaptoglobin.

The biosynthesis of rat haptoglobin, a hetrotetrameric glycoprotein (alpha 2 beta 2), requires the post-translational cleavage of its glycosylated primary translation product (prohaptoglobin) into alpha- and beta-subunits (Hanley, J. M., Haugen, T. H., and Heath, E. C. (1983) J. Biol. Chem. 258, 7858-7869). To elucidate the site(s) at which proteolytic cleavage occurs in prohaptoglobin, we have isolated a recombinant plasmid whose cDNA insert encodes for the carboxyl terminus of the alpha-subunit, the alpha beta-subunit junction, and the beta-subunit region, and also the entire 3'-untranslated region (142 base pairs) and poly(A) tail (55 base pairs) of the mRNA. A single arginine residue was found at the alpha beta-subunit junction region -Val-Gln-Arg-Ile-Ile-Gly-Gly-of prohaptoglobin. The sequence homology of this region with serine protease precursors suggests that post-translational processing of prohaptoglobin involves cleavage of the Arg-Ile bond and extraction of the Arg residue. The rat beta-subunit shows a high degree (approximately 80%) of sequence homology with its human counterpart although it possesses only two of the four N-glycosylation sites present in human haptoglobin beta-subunit.

Amino Acid Sequence↗
PubMed1984

Rat major acute-phase protein: biosynthesis and characterization of cDNA clone.

The major acute-phase protein (alpha 1-MAP) of rat serum is induced in response to inflammation. This induction may be attributed to a corresponding increase in the level of translatable mRNA for the protein. Using in vitro and in vivo systems, various biosynthetic processing intermediates of this glycoprotein have been isolated. alpha 1-MAP is translated in a rabbit reticulocyte system as a preprotein with an amino-terminal signal peptide and an apparent molecular weight of 51,000. Translation of rough microsomes yields a product with a mass of 57,000 Da, representing the core glycosylated form of alpha 1-MAP. Cotranslational glycosylation appears to occur in a stepwise fashion, since three glycosylated forms of alpha 1-MAP (51,000, 54,000, and 57,000 Da) were detected in polysome translations; these products were digested by endoglycosidase H to a 48,000-Da protein. Two intracellular forms of alpha 1-MAP were observed in vivo, a 57,000-Da (core carbohydrate sidechains) and a 66,000-Da protein (mature complex carbohydrate side-chains); the latter was the only component secreted into the culture medium. To extend our studies on this protein, a cDNA clone specific for alpha 1-MAP was isolated. The recombinant was positively identified by hybrid selection procedures and contains a 1.55-kb insert. Partial radiosequence analysis of the primary translation product indicated the distribution of Leu, Ile, Cys, and Met in the amino-terminal region of this protein. To relate the location of these amino acids with the nucleotide sequence, cDNA was analyzed by the method of Maxam and Gilbert. These results indicate that the cDNA insert contains the 3' poly(A) tail, and alignment of the 5' end of the cDNA with the available amino acid sequence of the primary translation product corroborated that the insert encodes the entire alpha 1-MAP protein except for the first four amino acids of the signal peptide.

Amino Acid Sequence↗
PubMed1983

Biosynthesis and processing of rat alpha 1-antitrypsin.

Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion.

Amino Acid Sequence↗
PubMed1981

The cyclic AMP-mediated induction of alkaline phosphatase in mouse L-cells.

A cell surface-localized glycoprotein that exhibits alkaline phosphatase activity was induced by treatment of mouse L-cell cultures with dibutyryl cyclic AMP. Treatment of cells with 1.5 mM dibutyryl cyclic AMP for a period of 7 days resulted in a approximately 2000-fold increase in the specific activity of the enzyme. Enzyme induction was dependent upon de novo RNA and protein biosynthesis since this induction was completely suppressed when actinomycin D (0.5 microgram/ml) or cycloheximide (5 microgram/ml) was administered with dibutyryl cyclic AMP. Further, the overall rates of incorporation of either [3H]glucosamine or [3H]leucine into macromolecules were identical in the presence or absence of dibutyryl cyclic AMP. Alkaline phosphatase was immunotitrated in 0.5% Triton X-100-solubilized cell extracts with antisera prepared against purified native enzyme and the results indicated that dibutyryl cyclic AMP stimulated that de novo synthesis of the enzyme. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis of specifically immunoprecipitated protein from cells incubated with either [35S]methionine or [6-3H]glucosamine demonstrated that dibutyryl cyclic AMP induced a 76,000-dalton glycoprotein that was characterized as alkaline phosphatase by its identity with native alkaline phosphatase that had been labeled with 32P in its active site. Electrophoretic analysis of specifically immunoprecipitated translation products from an in vitro protein-synthesizing system supplemented with L-cell RNA isolated from uninduced and cAMP-induced cells indicated that dibutyryl cyclic AMP induced the production of alkaline phosphatase-specific mRNA. These results suggest that dibutyryl cyclic AMP directly or indirectly influences the regulation of transcription of the alkaline phosphatase gene in L-cells.

Alkaline Phosphatase↗
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↗