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Biomedical subjects

J W Hamilton

Publications and source records attributed to J W Hamilton.

At least 127 records · Page 7Linked to original sources
PubMed1983

Thyroglobulin structure-function. The amino acid sequence surrounding thyroxine.

A 19-residue, thyroxine (T4)-containing peptide, Tryp-T4, has been isolated from the tryptic digest of a low molecular weight, iodine-enriched fragment derived from 19S bovine thyroglobulin. This tryptic peptide represents the only site of significant iodination in the parent polypeptide fragment. The amino acid sequence of the tryptic peptide has been determined and is NH2-Asn-Ile-Phe-Glu-T4-Gln-Val-Asp-Ala-Gln-Pro-Leu-Arg-Pro-Cys-Glu-Leu-Gln- Arg-COOH. The carboxyl-terminal sequence of this peptide shows a high probability of a beta-turn. These findings establish the involvement of at least a single unique sequence within thyroglobulin in thyroxine biosynthesis and the general nature of a hormonogenic site within this protein. This sequence contains at least 30% of the thyroxine present in 19 S bovine thyroglobulin.

Amino Acid Sequence↗
PubMed1983

Effects of isoproterenol and cycloheximide on parathyroid secretion.

Tissue slices or dispersed cells of bovine parathyroid gland were incubated with [3H]leucine to label the intracellular proteins and then tested for their secretory response to isoproterenol and cycloheximide at different calcium concentrations. Secretion of the newly synthesized as well as the older PTH and SP-I was stimulated by isoproterenol at all calcium levels tested, even when it was maximally enhanced by low calcium. Cycloheximide interfered with neither the secretory process nor the secretory response to different stimuli, but decreased the amount of PTH and SP-I secreted. We conclude that the inhibitor decreased the secretion by reducing the supply of PTH and SP-I. Calculations derived from the data reveal that, under most secretory conditions, newly synthesized PTH contributed a major portion of the total hormone secretion in bovine parathyroid cells.

Animals↗
PubMed1983

Development of basal and induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in the chicken embryo in ovo.

The development of the hepatic microsomal mixed-function oxidase system was studied to determine the basal level of embryonic enzyme activity and the inducibility of this system throughout growth and differentiation. Chicken embryo livers were assayed for basal and inducible hepatic aryl hydrocarbon hydroxylase (AHHase; designated elsewhere as AHH) activity from the first appearance of the liver as a discrete organ at 5 days of incubation (DI) through day 10 after hatching. In addition, whole-embryo and viscera preparations were assayed at 3 and 4 DI. Basal AHHase activity was equal to or greater than adult levels from 3 DI through hatching in all preparations (approximately 0.3-0.5 nmol/min per mg). A 3-fold increase in basal activity above adult values occurred at hatching. The onset of inducibility in chicken embryo liver between 5 and 6 DI was concomitant with hepatocyte differentiation. A developmental profile of 24-hr 3,4,3', 4'-tetrachlorobiphenyl-induced AHHase activity showed 15- to 30-fold induction over controls from 7 DI through day 10 after hatching, with a maximum of 15 nmol/min per mg at 14 DI and day 1 after hatching, a specific activity greater than 50% greater than maximal induction in the adult. Embryonic AHHase activity was also induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, beta-naphthoflavone, and sodium phenobarbital. Induction kinetics throughout embryonic development were similar to those reported for the adult chicken and other animals. These findings demonstrate development of a mixed-function oxidase system in very early embryogenesis and then in the liver as it differentiates. Liver AHHase activity is inducible throughout development and perinatally but such activity is under strict developmental regulation. The chicken embryo has adult levels of AHHase activity which would be sufficient to achieve metabolic activation of promutagens/carcinogens before and after hepatocyte differentiation.

Animals↗
PubMed1983

Carnitine transport in rat small intestine.

Although L-carnitine has been given orally to patients with systemic carnitine deficiency with successful control of the disease and is present in a variety of dietary sources, there is little available information on the physiology of its absorption. We therefore studied intestinal carnitine absorption in the rat by measuring the uptake of radioactive L-carnitine by everted intestinal rings and sacs. Active transport was demonstrated in duodenum and jejunum, but not ileum, with intracellular concentrations higher than medium concentrations at steady state and by the prevention of concentration gradients with anoxia, metabolic inhibitors, and replacement of sodium ion. Studies of the relationship of uptake to carnitine concentration demonstrated the presence of two components of transport: a saturable component (with a Km of between 206 and 316 microM) that could be inhibited by the metabolically inactive D-isomer and by acetylcarnitine and a linear component that we presume represents diffusion.

Anaerobiosis↗
PubMed1983

The content of carboxyl-terminal fragments of parathormone in extracts of fresh bovine parathyroids.

Fresh parathyroid gland homogenates and fractions thereof were analyzed for their content of PTH and carboxyl-terminal fragments of the hormone. The tissue proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then extracted from gel fractions for RIA. Native PTH and PTH-(37-84) were used as standards to mark the migration positions of these peptides in the gels. The RIA for carboxyl-terminal hormone fragments used PTH-(37-84) as radioiodinated tracer and responded equally on a molar basis to either PTH or PTH-(37-84), making possible quantitative evaluation of both peptides in one assay after their separation. The results indicated that tissue homogenates contain 0.3-0.5 PTH-(37-84) moleq for each mole of PTH. Particulate fractions of the homogenates contained 0.15-0.3 moleq of fragment/mol PTH, while the high speed supernatant fraction of the homogenate contained about 2 moleq of fragment/mol PTH. When the experiments were performed using homogenization and fractionation buffers that contained numerous protease inhibitors, the ratios of carboxyl-terminal PTH fragment to intact hormone were not decreased, indicating that the hormone fragments were not produced during tissue processing. In addition, PTH added to tissue homogenates was not degraded during subsequent manipulations. The results demonstrate that fresh bovine parathyroid tissue contains substantial levels of carboxyl-terminal PTH peptide fragments, which can be measured by RIA after separation from PTH and other hormonal species. The data support the hypothesis that hormone fragments reside in regions of the cell different from those that contain PTH.

Animals↗
PubMed1983

Cleavage of parathyroid hormone to the 1-34 and 35-84 fragments by cathepsin D-like activity in bovine parathyroid gland extracts.

We have obtained a crude enzyme preparation from bovine parathyroid gland homogenates which when incubated with PTH, cleaves the hormone into two major fragments. Isolation and chemical analysis has led to the identification of these peptides, the 1-34 fragment and the 35-84 fragment. Digestion of PTH was totally inhibited by the inclusion of the cathepsin D inhibitor, pepstatin, in the enzyme digest. A comparison of the digest obtained using the crude enzyme fraction vs. digestion of PTH by purified bovine cathepsin D led to the findings that the same peptide products were formed in each case. The natural 1-34 hormone fragment derived from the procedure has been determined to be fully biologically active in a bone resorption system.

Amino Acids↗
PubMed1982

Amino acid sequence of rat alpha-lactalbumin: a unique alpha-lactalbumin.

The amino acid sequence of rat alpha-lactalbumin has been determined. Unlike other alpha-lactalbumins which contain 122 or 123 amino acids, rat alpha-lactalbumin is unique in that it contains 140 amino acids. The extra amino acids are a 17 amino acid extension at the carboxyl terminus. The amino acid sequence of this extension is Gly124-Ala-Pro-Ala-Leu-Val-Val130-Pro-Ala-Leu-Asp-Gly135-Glu-Thr-Pro-Val-Pro140 . The extension is proline rich, which may contribute to the anomalous structural properties of rat alpha-lactalbumin. The amino acid sequence from residues 1 to 123 is similar to that of other alpha-lactalbumins. One possible explanation for the 17 amino acid extension is a mutation at the termination codon.

Amino Acid Sequence↗
PubMed1982

Similarity of secretory protein I from parathyroid gland to chromogranin A from adrenal medulla.

We have compared the amino acid and carbohydrate compositions, partial amino acid sequences, immunological crossreactivity, and physical properties of secretory protein I of the parathyroid gland and chromogranin A of adrenal gland. This comparison indicates that these proteins are similar molecules. Because secretory protein I is present in secretory granules containing parathormone and is cosecreted with the hormone, and because chromogranin A is contained within chromaffin granules and, likewise, is secreted with the catecholamines, the present observations raise the possibility that this class of protein plays a general role in hormone secretion or storage mechanisms.

Adrenal Medulla↗
PubMed1981

Isolation and partial characterization of secretory protein I from bovine parathyroid glands.

Secretory protein I, a protein that is cosecreted with parathormone, has been isolated from bovine parathyroid tissue. The purification procedure was aided by the inclusion in the starting material of fresh tissue that had been incubated with radioactive amino acids to label the newly formed secretory protein I. The isolation of the secretory protein I was then followed by locating the radioactive species. Later, purification was also followed by radioimmunoassay. The procedures included salt fractionation, gel filtration, and two steps of ion-exchange chromatography, yielding a 96-fold purification of secretory protein I. The final product contained two species that were shown to be related by comparison of their tryptic peptides and the release of only a single major residue at each step of the Edman degradation. On the basis of amino acid analysis, secretory protein I contains about 30% acidic amino acid residues, contributing to an isoelectric point of 4.5, and has a minimum molecular weight of about 70 000. It contains 2.6% carbohydrate. A radioimmunoassay was established for secretory protein I. A partial amino acid sequence spanning the first 32 residues of the amino-terminal region was obtained. This portion of the structure appeared to be unrelated to those of the known parathyroid hormonal peptides.

Amino Acid Sequence↗
PubMed1981

Isolation, structure and synthesis of a heptapeptide with in vitro ACTH-releasing activity from porcine hypothalamus.

Significant CRF activity was found in a fraction with Rf = 0.82-0.7 or VE/VT = 0.41-0.48 obtained by gel filtration of acid extracts of pig hypothalami on Sephadex G-25. The activity of this fraction decreased markedly during subsequent purification, particularly in the last two steps. From this fraction, a heptapeptide with significant ACTH releasing activity in vitro, was isolated in pure state, and its amino acid sequence was established as H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH. This heptapeptide was synthesized by solid phase methods. The CRF activity of synthetic heptapeptide in vitro was low but could be potentiated by a cofactor fraction from rat hypothalamic extract.

Adrenocorticotropic Hormone↗
PubMed1981

Cloning and sequence of several alpha 2u-globulin cDNAs.

We describe a simple cloning procedure for alpha 2u-globulin that requires neither enrichment of mRNA for cloning nor purification of a specific probe for screening recombinant colonies. Total adult male liver poly(A)+RNA was used as template for cloning, and the subsequent recombinant colonies were screened by comparing hybridization to radioactive cDNA probes prepared from hepatic male and female mRNA, respectively. Almost all of the selected "male-specific" clones were later shown to contain alpha 2u-globulin sequences. This cloned alpha 2u-globulin cDNA has been shown to specifically hybridize to male rat liver RNA, which, when isolated and translated in vitro, codes for a 21,000-dalton protein (pro-alpha 2u-globulin) immunologically identical to alpha 2u-globulin. When translation occurs in the presence of pancreatic microsomes this in vitro synthesized pro-alpha 2u-globulin is processed to the 19,000-dalton mature form of alpha 2u-globulin. The nucleotide sequence of the alpha 2u-globulin cDNA has been determined, thus elucidating the complete amino acid sequence of alpha 2u-globulin and most of the hydrophobic "leader" sequence of pro-alpha 2u-globulin. The amino acid sequence deduced from the cDNA is in agreement with the partial sequence that we previously determined by sequential Edman degradation of the purified protein. alpha 2u-Globulin cDNA clones contain within the 3'-untranslated region one or both of the two putative polyadenylylation/transcription termination sites (A-A-T-A-A-A and A-A-T-T-A-A-A). Either of these can be used, generating alpha 2u-globulin mRNA species of two lengths. A codon usage analysis of the cDNA showed that, although all six leucine codons are used for the 14 leucine residues in mature alpha 2u-globulin, the seven leucines in the partial leader sequence reported are all encoded by the same codon, CTG. The primary amino acid sequence contains a unique Asn-Gly-Ser sequence, likely to be in beta-turn conformation, as the probable site of glycosylation for this glycoprotein.

Alpha-Globulins↗
PubMed1981

Sequence homology in the amino-terminal and active-site regions of thermolabile glyceraldehyde-3-phosphate dehydrogenase from a thermophile.

The unusual thermolability of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU (Crabb et al., Biochemistry 16:4840-4847, 1977) has provided the first opportunity to study a homologous enzyme from the same genus that exhibits a marked difference in thermostability. In pursuit of the structural bases for the thermostability of proteins, the sequences of the amino terminus (residues 1 through 27) and the active-site cysteine cyanogen bromide peptide (residues 130 through 167) of this enzyme have been determined and compared with sequences of the enzyme from other sources. The importance of comparing phylogenetically related proteins is evident from the 87% identity found between these sequences in the enzyme from B. coagulans and Bacillus stearothermophilus, versus only 45% identity for all other known sequences. The marked sequence identity of the enzyme from the two Bacillus species drew attention to the variable region (residues 138 through 140a) which is exposed to the exterior of the quaternary structure of this enzyme. Based on the reported crystallographic structures of the enzyme from lobster muscle and B. stearothermophilus and space-filling models of the variable region, the segment Asp-Pro-Lys-Ala in B. stearothermophilus should be more thermostable than the analogous sequence, Asp-Ala-Ala-Asn, from B. coagulans. In addition, the space-filling models suggested that the spatial relationship of an amino acid side chain and its potential for close packing and interactions with neighboring side chains may be more important than the type of amino acid substituted.

Amino Acid Sequence↗
PubMed1980

Synthesis, intracellular distribution, and secretion of multiple forms of parathyroid secretory protein-I.

Examination of whole cell extracts and subcellular fractions of dispersed porcine parathyroid cells incubated with [35S]methionine indicates that two species of secretory protein-I, 72,000 and 64,000 daltons, respectively, are synthesized. Two secretory protein-I species of molecular weights equivalent to those in the cell but with slightly different isoelectric points were secreted; calcium suppressed the secretion of both of these. The secretory protein-I of cell and medium were shown to be related to each other and to previously identified secreted secretory protein-I by comparison of their 35S-labeled tryptic peptides and location of methionine in positions 7, 15, and 32 of the peptide chains. Both of the cellular species appeared to be enclosed within membranes similar to those containing parathyroid hormone and its immediate biosynthetic precursor because they were associated with the membrane fraction of the cell, were not digested when the membranes were exposed to trypsin, and were extracted from these membranes, as were parathyroid hormone and proparathyroid hormone, with dilute sodium deoxycholate. We did not find an amino-terminal precursor form of secretory protein-I in an incubation as short as 2 min with [35S]methionine, whereas [35S]proparathyroid hormone was readily detected, indicating that processing of secretory protein-I involves a direct conversion of the pre-protein to the secretory protein-I. Posttranslational glycosylation or deletion of carboxy-terminal region of the secretory protein-I species might account for the differences in molecular weights and isoelectric points of the cellular and secreted forms.

Animals↗