Biomedical subjects

J E Shively

Publications and source records attributed to J E Shively.

At least 163 records · Page 9Linked to original sources

PubMed1987

Primary structure of apolipoprotein A-II from inbred mouse strain BALB/c.

The primary structure of apolipoprotein A-II (apoA-II) isolated from the plasma high density lipoprotein (HDL) fraction of the inbred mouse strain BALB/c is described in this work. The complete 78 amino acid protein sequence was determined by proteolytic fragmentation, gas-phase microsequence analysis, and fast atom bombardment (FAB) mass spectrometry. The apolipoprotein has a calculated molecular weight of 8,715 and a net negative charge conveyed by ten acidic and eight basic amino acid residues. There exists a 55% amino acid sequence homology between the BALB/c mouse apoA-II and human apoA-II. Unlike human plasma apoA-II, which exists as a disulfide dimer, BALB/c apoA-II lacks cysteine and is a monomer. BALB/c apoA-II contains one residue each of histidine and arginine, neither of which are found in the human A-II protein. Chou and Fasman analysis of the BALB/c apoA-II primary structure predicts approximately 68% alpha-helical potential compared with a 62% potential for human apoA-II. The alpha-helical domains are structurally amphipathic, generating a polar and an apolar face consistent with the proposed models describing apolipoprotein-phospholipid interaction.

Amino Acid Sequence↗

PubMed1987

Partial amino acid sequence analyses of human placental insulin receptor.

Tryptic peptides were isolated from human insulin receptor (1.8 nmol) by sequential reverse-phase HPLC on SynChropak RP-C8 and Vydac C-18. Nearly 20 peptides were analyzed for amino acid composition, which revealed that the yield of tryptic peptides averaged 300 pmol. By gas-phase microsequencing five peptides were sequenced. N-acetylglucosamine was found in two peptides. These data are compared with the amino acid sequence of the insulin receptor deduced from cloned cDNA encoding for human insulin receptor.

Amino Acid Sequence↗

PubMed1986

New molecular forms of cholecystokinin. Microsequence analysis of forms previously characterized by chromatographic methods.

Cholecystokinin previously has been characterized by chromatographic and immunological techniques. Analysis of cDNA has revealed the structure of preprocholecystokinin. The predicted processing sites of preprocholecystokinin cannot account for the multiple forms of cholecystokinin detected. This report details the isolation and characterization of cholecystokinin peptides containing 58, 39, 33, 25, 18, 8, 7, and 5 amino acids. None of the cleavages that occurred to form these peptides was at the carboxyl side of double basic residues. Cholecystokinin 25, 18, and 7 have not previously been isolated and identified by sequence analysis. The processing that forms these peptides includes cleavage after single basic residues for the 58, 39, 33, and 8 amino acid peptides. The 25, 18, 7, and 5 amino acid peptides must be formed by other endopeptidases or combinations of endo- and exopeptidases. The analysis of this series of peptides provides the chemical basis for structural differences in cholecystokinin molecules previously demonstrated by chromatographic and immunological methods. These structures provide insight into tissue-specific processing that occurs for this important regulatory peptide in intestine and brain.

Amino Acid Sequence↗

PubMed1986

Complete amino acid sequence of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes.

The complete amino acid sequence of porcine hepatic microsomal NADPH-cytochrome P-450 reductase has been determined by microsequence analysis on several sets of proteolytic fragments. Sequence studies were performed initially on a 20-kilodalton (kDa) fragment and then on 80-kDa fragment. The amino-terminal end of the mature protein was blocked with an acetyl group, followed by 676 amino acid residues. It has been revealed that the COOH-terminal 20-kDa fragment has been derived from original enzyme by cleavage at the Asn-Gly (residues 502-503) linkage by an unknown mechanism. An NADPH-protected cysteine residue is located at residue 565, near a region exhibiting high sequence homology with ferredoxin-NADP+ reductase. The FMN and FAD binding regions are possibly located in the amino-terminal region and the middle part of the protein molecule, respectively, as suggested by Porter and Kasper [Porter, T. D., & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 973-977]. When this sequence is compared with that of rat enzyme, 60 amino acid residues are substituted, probably due to species differences. However, total sequence homology between these enzymes is 90%. Hydropathy plot analysis reveals that two regions from residues 27-43 and from residues 523-544 exhibit a high degree of hydrophobicity, suggesting membrane binding or interaction with cytochrome P-450.

Amino Acid Sequence↗

PubMed1986

Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase.

We have isolated cDNA clones of the mRNA for rat UDP-glucuronosyltransferase that catalyzes the glucuronidation of 4-nitrophenol, by using synthetic oligonucleotides as hybridization probes. The complete nucleotide sequence of the 1,927-base pairs cDNA insert has been determined. With untranslated sequences of 124 and 216 base pairs in the 5'- and 3'-terminal regions, respectively, the cDNA insert contained 1,587 base pairs that encode a complete primary structure of a putative precursor form of 4-nitrophenol UDP-glucuronosyltransferase with a calculated molecular weight of 60,114. The cDNA sequence also indicates the presence of 25 amino acids preceding the sequence determined by microsequence of the isolated protein. This extrapeptide, for the most part, consists of hydrophobic amino acids which are characteristic of the signal peptides as found for secretory proteins and most transmembrane proteins. Furthermore, the deduced amino acid sequence contains a putative halt transfer signal of a hydrophobic segment (residues 487-510), which is flanked on both sides by the peptide segments of highly charged amino acid residues (residues 463-486 and 511-529). These features are consistent with the properties of transmembrane proteins. Specific cDNA probes were used to analyze the induction of the enzyme in rat tissues by treatment with 3-methylcholanthrene. RNA blot analysis showed that 3-methylcholanthrene increased 10- to 15-fold the amount of hybridizable mRNA in liver. The livers and kidneys from 3-methylcholanthrene-treated rats were found to contain almost the same amount of hybridizable mRNA, although the basal level in the kidney was much higher than that of the liver, and the amounts in the lung were much lower than that of the liver and kidney.

Amino Acid Sequence↗

PubMed1986

Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase.

Through the use of monospecific antibodies directed against hepatic cytochrome P-450j, an enzyme induced in rats treated with ethanol or isoniazid, we have purified from human liver the related cytochrome P-450 termed HLj. HLj resembles rat P-450j and P-450 LM3a, the homologous cytochrome in rabbit liver, in its NH2-terminal amino acid sequence, in being in highest concentration in liver microsome samples prepared from two patients intoxicated by ethanol and one patient given isoniazid, and in catalyzing the metabolic activation of the procarcinogen N-nitrosodimethylamine. Furthermore, each of nine human liver RNA samples contained a species of mRNA hybridizable to a cloned HLj cDNA. We conclude that HLj is related by structure, function, and some regulatory characteristics to rat P-450j and rabbit P-450 LM3a, cytochromes critical for metabolism of several clinically relevant cytotoxic and carcinogenic agents.

Adult↗

PubMed1986

Identification of the arylazido-beta-alanyl-NAD+-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry.

We have identified the site labeled by arylazido-beta-alanyl-NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+) in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry. This NAD+ photoaffinity analogue has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in a very specific manner and probably at the active site of the enzyme [Chen, S., Davis, H., Vierra, J. R., & Guillory, R. J. (1984) Biochem. Biophys. Stud. Proteins Nucleic Acids, Proc. Int. Symp., 3rd, 407-425]. The label is associated exclusively with a tryptic peptide that has the sequence Ile-Val-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue at position seven was predominantly labeled and suggested to be the site modified by arylazido-beta-alanyl-NAD+. This cysteine residue corresponds to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity. The present investigation clearly demonstrates that arylazido-beta-alanyl-NAD+ is a useful photoaffinity probe to characterize the active sites of NAD(H)-dependent enzymes.

Affinity Labels↗

PubMed1986

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone.

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.

Acetophenones↗

PubMed1986

Chemical modification and inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone.

The alkylating agent 2-bromo-4'-nitroacetophenone (BrNAP) binds covalently to each of 10 isozymes of purified rat liver microsomal cytochrome P-450 (P-450a-P-450j) but substantially inhibits the catalytic activity of only cytochrome P-450c. Regardless of pH, incubation time, presence of detergents, or concentration of BrNAP, treatment of cytochrome P-450c with BrNAP resulted in no more than 90% inhibition of catalytic activity. Alkylation with BrNAP did not cause the release of heme from the holoenzyme or alter the spectral properties of cytochrome P-450c, data that exclude the putative heme-binding cysteine, Cys-460, as the major site of alkylation. Two residues in cytochrome P-450c reacted rapidly with BrNAP, for which reason maximal loss of catalytic activity was invariably associated with the incorporation of approximately 1.5 mol of BrNAP/mol of cytochrome P-450c. Two major radio-labeled peptides were isolated from a tryptic digest of [14C]BrNAP-treated cytochrome P-450c by reverse-phase high performance liquid chromatography. The amino acid sequence of each peptide was determined by microsequence analysis, but the identification of the residues alkylated by BrNAP was complicated by the tendency of the adducts to decompose when subjected to automated Edman degradation. However, results of competitive binding experiments with the sulfhydryl reagent 4,4'-dithiodipyridine identified Cys-292 as the major site of alkylation and Cys-160 as the minor site of alkylation by BrNAP in cytochrome P-450c.

Acetophenones↗

PubMed1986

Two plasma fibronectin fragments with different gelatin-binding properties.

Two subtilisin-generated fragments of human plasma fibronectin exhibit significantly different gelatin affinities. One fragment of 60 kdaltons elutes from gelatin-Sepharose, after intact fibronectin, with pH 5.5, 50 mM citrate, 0.1 M NaCl. A 40 kdalton fragment, which can be derived from the 60 kdalton fragment, remains bound to gelatin under the same conditions. Furthermore, upon urea gradient elution, the 60 kdalton fragment dissociates from gelatin before the smaller fragment. Of the two fragments, the 60 kdalton fragment also demonstrates affinity for heparin. Identical amino-terminal sequences for the two fragments indicate that the heparin-binding activity resides in the difference region at the carboxyl-terminal of the 60 kdalton fragment.

Amino Acid Sequence↗

PubMed1986

Isolation and structure of the principal products of preproglucagon processing, including an amidated glucagon-like peptide.

The principal products derived from in vivo processing of anglerfish preproglucagon II were isolated and their structures determined. The structures were confirmed by a combination of automated Edman degradation, amino acid analysis, and fast atom bombardment mass spectrometry. The peptide corresponding to anglerfish preproglucagon II-(22-49) (numbering from the amino terminus of preproglucagon) was isolated intact and defines the site of signal cleavage to be between Gln-21 and Met-22. Glucagon from the anglerfish preproglucagon gene II was found to correspond to preproglucagon II-(52-80) (numbering from the amino terminus). Three forms of a glucagon-like peptide derived from preproglucagon II were also isolated. The structure of the longest form was consistent with the sequence of preproglucagon II-(89-122) deduced from the cDNA, His-Ala-Asp-Gly-Thr-Tyr-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gln-Asp-Gln-Ala- Ala-Lys-Asp-Phe-Val-Ser-Trp-Leu-Lys-Ala-Gly-Arg-Gly-Arg-Arg-Glu. The carboxyl-terminal portion deduced from the cDNA remains intact in this form. A second form, preproglucagon II-(89-119) appears to result from proteolytic processing of the major form at the two adjacent arginine residues occurring at the carboxyl terminus. This second form has a glycine residue at its carboxyl terminus and is processed to the third form (preproglucagon II-(89-118)) which contains a carboxyl-terminal arginineamide. Radiolabeling studies in primary tissue culture support the observation that glucagon (preproglucagon II-(52-80], preproglucagon II-(89-122), and preproglucagon II-(89-119) are products of proglucagon processing in vivo.

Amino Acid Sequence↗

PubMed1986

The amino acid sequence of kinetensin, a novel peptide isolated from pepsin-treated human plasma: homology with human serum albumin, neurotensin and angiotensin.

A novel nonapeptide with neurotensin-like immunoreactivity was isolated from pepsin-treated human plasma by dialysis, ion-exchange chromatography and high performance reversed-phase liquid chromatography. The amino acid sequence was determined by automated gas-phase sequence analysis as Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe-Leu. Sequence homology with human serum albumin and with the biologically active peptides neurotensin and angiotensin is demonstrated. The name proposed for this peptide is kinetensin.

Amino Acid Sequence↗

PubMed1986

Amino terminal sequence analysis of human placenta aromatase.

The amino acid composition and the amino-terminal amino acid sequence from position 1 to 21 of human placenta aromatase were determined. In addition, a cysteine containing peptide with a sequence homologous to those of peptides containing the cysteine residue which was suggested to provide the proximal thiolate ligand to the heme in other cytochrome p-450 isozymes, was identified. The results indicate that aromatase is a cytochrome p-450 protein, probably derived from a new cytochrome p-450 family.

Amino Acid Sequence↗

PubMed1986

The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 11 beta-, 18-hydroxylation, and aldehyde synthesis.

In order to establish the nature of the aldosterone synthetase activity in the adrenal cortex, we have used porcine adrenal, bovine adrenal cortex, highly purified bovine and porcine 11 beta-/18-hydroxylase, and antibodies raised against the latter enzyme. Mitochondria from two zones (glomerulosa and fasciculata) of the bovine cortex synthesize aldosterone, but those from glomerulosa are much more active than those from fasciculata. Partially purified (cholate-extracted plus ammonium sulfate-precipitated) extracts of mitochondria from the two zones are equally active in catalyzing all three steps in the conversion of 11-deoxycorticosterone to aldosterone. 18-Hydroxylase and aldehyde synthetase activities (18-hydroxycorticosterone----aldosterone) were completely precipitated from cholate extracts of mitochondria from bovine adrenal by antibodies to the pure porcine enzyme. No activity corresponding to any of the three steps in the conversion of 11-deoxycorticosterone to aldosterone was found in extramitochondrial fractions of the bovine cortex. Synthesis of aldosterone by the pure porcine enzyme was inhibited by antibodies to this enzyme and by metyrapone (an inhibitor of 11 beta-/18-hydroxylase). When fractions of porcine adrenal, resulting from purification of the enzyme from mitochondria, were exhaustively tested for any of the enzyme activities required for the synthesis of aldosterone, activity was found only in those fractions containing the 11 beta-/18-hydroxylase, i.e. no additional enzyme was discarded during the purification procedure. It is concluded that the only adrenocortical enzyme capable of synthesizing aldosterone in bovine and porcine adrenal is the well known 11 beta-hydroxylase, that the conversion of 18-hydroxycorticosterone to aldosterone is catalyzed by this cytochrome P-450, and that this step (aldehyde synthetase) requires the heme of the P-450 as demonstrated by the photochemical action spectrum.

Adrenal Cortex↗

PubMed1986

Isolation and primary structure of the califins, three biologically active egg-laying hormone-like peptides from the atrial gland of Aplysia californica.

The atrial gland of the marine mollusk Aplysia californica contains several biologically active peptides that are thought to be important in reproductive function. In the present study, three novel peptides, which we named califin A, B, and C, were purified from extracts of atrial glands by high performance liquid chromatography, and their primary structures were determined. Each consists of a 36-residue subunit bound by a single disulfide bond to an 18-residue subunit. The large subunits differ from each other by one or two residues, whereas the small subunits are identical. The large subunits are 78-83% homologous to egg-laying hormone (ELH), a 36-residue peptide synthesized by the neuroendocrine bag cells of Aplysia. Like ELH, the califins excite LB and LC cells of the abdominal ganglion and cause egg laying when injected into sexually mature animals. Based on previously described DNA sequence data, each califin is likely to be derived from one of several precursor proteins that are encoded by members of the ELH gene family. Califin A is encoded on the peptide A precursor, and califin B may be encoded on the peptide B precursor. No gene encoding califin C has been sequenced. Because peptides A and B are also biologically active, the precursors encoding them and califins A and B are polyproteins. The possible role of atrial gland peptides as pheromones is discussed.

Amino Acid Sequence↗

PubMed1986

The effect of tumor CEA content and tumor size on tissue uptake of indium 111-labeled anti-CEA monoclonal antibody.

This study was undertaken to determine the effect of tumor size and tumor carcinoembryonic antigen (CEA) content on the uptake of indium 111 (111In)-labeled anti-CEA monoclonal antibody in nude mice bearing xenografts. The tumor cell lines were WiDr, SW403, and LS174T, human colon cancer derivatives. The murine breast carcinoma cell line EMT-6 was used as a control. Tumor CEA levels (ng/g of tumor +/- standard error of the mean [SEM], measured by enzyme immunoassay (EIA) were: EMT-6, 0; WiDr, 105 +/- 5.7; LS174T, 2052 +/- 198; SW403, 17,575 +/- 1,785. The 111In-labeled monoclonal antibody was injected intravenously into mice bearing a single tumor. At 48 hours postinjection, scintiscan was performed, and the mice were killed so that biodistribution studies could be performed. The uptake of the monoclonal antibody was expressed as percent injected counts per minute per gram of tissue +/- SEM. The non-CEA-producing tumor, EMT-6, showed the lowest tumor uptake (1.4 +/- 0.3). WiDr, an intermediate CEA-producing tumor, showed some tumor uptake (16.4 +/- 1.5). The high CEA-producing tumors, SW403 and LS174T, had high tumor uptake (29.5 +/- 5.0 and 51.1 +/- 6.1, respectively). Biodistribution and scintiscan quality were closely related. Although LS174T had the best tumor uptake, SW403 had the highest CEA tumor content, indicating tumor CEA content cannot entirely predict scintiscan and biodistribution results. Tumor-to-blood (T/B), tumor-to-liver (T/L), and liver-to-blood (L/B) ratios were calculated for each animal and compared with tumor size. It was found that T/L had a negative correlation with tumor size (r = -0.72) and L/B had a positive correlation with tumor size (r = 0.94). These ratios may be useful clinically to follow response to therapy.

Animals↗

PubMed1986

Imaging of human colorectal adenocarcinoma with indium-labeled anticarcinoembryonic antigen monoclonal antibody.

Twenty patients with 21 primary colorectal adenocarcinomas were studied with 2 mCl (7.6 X 10(7) becquerels) of indium-labeled monoclonal antibody (200 micrograms) specific for carcinoembryonic antigen (CEA). Fifteen lesions (71%) were visualized by gamma camera scintigraphy at 48 hours postinjection. Tumors that were identified by immunoscintigraphy were large (38.10 +/- 17.76 cm3 vs 6.00 +/- 1.65 cm3), had a grossly fungating component, had a high content of CEA by enzyme immunoassay (12.9 +/- 3.6 micrograms/g vs 3.3 +/- 1.7 micrograms/g), and had an apical and/or intraluminal staining pattern on immunohistologic section. Patients whose tumors were visualized had a low serum CEA level (1.9 +/- 0.4 ng/mL vs 14.6 +/- 8.0 ng/mL). Prospective selection of patients for follow-up imaging or therapy with radiolabeled monoclonal antibodies may be feasible using these measurements.

Antibodies, Monoclonal↗

PubMed1986

The primary structure of cytochrome P-450d purified from rat liver microsomes: prediction of helical regions and domain analysis.

The complete amino acid sequence of rat liver microsomal cytochrome P-450d (induced by isosafrole) was deduced by microsequence analysis of the tryptic peptides after separation by reverse-phase HPLC and alignment by comparison to the cDNA sequence reported by K. Kawajiri, O. Gotoh, K. Sogawa, Y. Tagashira, M. Muramatsu, and Y. Fujii-Kuriyama (1984, Proc. Natl. Acad. Sci. USA 81, 1649-1653). Results from the two approaches are in complete agreement with the exception of two residues, Lys-30 (Arg in cDNA) and Phe-261 (Ser in cDNA). As previously reported by us (L. H. Botelho, D. E. Ryan, P.-M. Yuan, R. Kutney, J. E. Shively, and W. Levin (1982, Biochemistry 21, 1152-1155) the NH2-terminal sequence of the mature protein lacks the NH2-terminal Met residue. Comparison of the rat cytochrome P-450d sequence with the mouse cytochrome P3-450 cDNA sequence reported by S. Kimura, F.J. Gonzalez, and D.W. Nebert (1984, Nucl. Acids Res. 12, 2917-2928) reveals a high sequence homology with a total of 32 amino acid differences including six conferring charge changes. Prediction of the secondary structure of cytochrome P-450d yields a maximum of 17 helices, two of which may be poly(Pro)-like helices adjacent to potential membrane-spanning alpha-helices. Four of the alpha-helices are sufficiently hydrophobic to traverse the endoplasmic reticulum. The remaining helices are largely amphiphilic. Analysis of the helices in reference to predicted membrane topology suggests that cytochrome P-450d either has one large and one small globular domain separated by a transmembrane domain and anchored by NH2-terminal and COOH-terminal transmembrane domains, or has one large globular domain anchored at both ends by transmembrane domains.

Amino Acid Sequence↗