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J T Potts

Publications and source records attributed to J T Potts.

At least 19 recordsLinked to original sources
PubMed1979

Cellular processing of pre-proparathyroid hormone involves rapid hydrolysis of the leader sequence.

The cellular synthesis of parathyroid hormone (PTH) involves two consecutive cleavages of NH2-terminal peptide sequences from a larger precursor, pre-proparathyroid hormone (Pre-ProPTH). The initial cleavage consists of the removal of an NH2-terminal leader sequence either during or shortly after biosynthesis of the polypeptide chain is complete. To determine the fate of the cleaved leader sequence, we prepared, by chemical synthesis, a peptide based on the known structure of the leader sequence of pre-proparathyroid hormone and used this peptide labeled with 125iodine as a marker to monitor the recovery of the putative cellular leader peptide during extraction and electrophoresis of [35S]methionine-labeled proteins from pulse-labeled parathyroid gland slices. Under conditions in which the recovery of the synthetic leader peptide was 50 to 70%, we found no detectable 35S-labeled product in the region of sodium dodecyl sulfate gels where the synthetic peptide migrates. In view of the known methionine content of pre-proparathyroid hormone and proparathyroid hormone (ProPTH), it would have been possible to detect endogenously labeled leader peptide if present in amounts equal to 0.05% of the amount of labeled ProPTH present in the tissues. These observations indicate that the cellular conversion of Pre-ProPTH to ProPTH involves a rapid hydrolysis of the leader peptide either during or immediately after its removal from the precursor.

Animals↗
PubMed1979

Isolation and identification of previtamin D3 from the skin of rats exposed to ultraviolet irradiation.

The process of the photolytic activation of vitamin D precursor(s) in the skin has been elucidated by a detailed analysis of the products formed after ultraviolet light exposure. The photolytic product isolated from the skin of rats exposed to ultraviolet irradiation was identified as previtamin D3 by several criteria including its (a) characteristic ultraviolet absorption spectrum, (b) mass spectrum, and (c) thermal isomerization to vitamin D3, which itself was identified also by mass spectroscopy. Vitamin D3 per se was not formed by ultraviolet irradiation--vitamin D3 arises exclusively from the thermal conversion of previtamin D3. Detectable amounts of lumisterol3 or tachysterol3 were not seen.

Animals↗
PubMed1979

Cloning and nucleotide sequence of DNA coding for bovine preproparathyroid hormone.

We have cloned in Escherichia coli a DNA copy of mRNA coding for bovine preproparathyroid hormone. Double-stranded DNA was inserted into the Pst I site in plasmid pBR322 by using the poly(dG)-poly(dC) homopolymer extension technique to join the DNA molecules. Recombinant plasmids coding for preproparathyroid hormone were identified by the plasmid's ability to arrest specifically the translation of preproparathyroid hormone mRNA. The nucleotide sequence of the largest recombinant was determined by using both chemical and enzymatic techniques. The parathyroid insert contains 470 nucleotides--102 nucleotides from the 5' noncoding region of the mRNA, 345 nucleotides representing the entire coding region, and 23 nucleotides from the 3' noncoding region. The coding sequence clarifies the hormone's amino acid sequence, which has been disputed. Codon usage is discussed.

Amino Acid Sequence↗
PubMed1979

Subcellular distributions of parathyroid hormone, hormonal precursors, and parathyroid secretory protein.

Distributions of parathyroid hormone (PTH), proparathyroid hormone (ProPTH), preproparathyroid hormone (PreProPTH), and parathyroid secretory protein (PSP) were analyzed in subcellular fractions prepared from homogenates of bovine parathyroid glands. Slices of bovine parathyroid glands were incubated with radiolabeled amino acids for 3--30 min to selectively label newly synthesized proteins. Subcellular fractions were prepared from homogenates of the gland slices by differential centrifugation. Newly synthesized labeled hormonal polypeptides in the fractions were analyzed by electrophoresis on polyacrylamide gels, and total amounts of PTH and ProPTH (previously formed and newly synthesized) were determined by immunoassay. Ninety percent of total immunoreactive, 70--80% of newly synthesized PTH, ProPTH, and PreProPTH, and 50% of PSP were found in sedimentable particulate fractions. The low speed (800 X g) pellet, which consisted predominantly of cell debris and nuclei with adherent remnants of cytoplasm, contained 30--50% of the ProPTH and PTH. The intermediate speed (10,000 X g) pellet, which contained granules, was relatively enriched in PTH. Most particulate-associated hormone could be solubilized by treatment with deoxycholate (DOC) 98% and 97% of radiolabeled and 93% and 83% of immunoreactive ProPTH and PTH, respectively, in particulates sedimenting at 10,000 and 105,000 X g were rendered DOC-soluble. Approximately 50% of the PTH and ProPTH in the particulates resisted digestion by combined trypsin and chymotrypsin, whereas PreProPTH was completely susceptible to proteolysis. Up to 50% of the radiolabeled PTH and ProPTH added exogenously to parathyroid gland slices before homogenization became associated with the particulate fractions, and 70--80% or radiolabeled PreProPTH added to the subcellular fractions readily associated with the sedimentable material. The results indicate that in homogenates of parathyroid glands, PTH, ProPTH, PreProPTH, and PSP are associated with particulate structures. Furthermore, up to 50% of the association of ProPTH, PTH, and PSP with particulate fractions seems to be nonsepcific and occurs during the disruption of the tissues. The remaining 50% or more of hormonal protein is presumably sequestered within membrane-limited structures, such as microsomal vesicles. The complete susceptibility in particulate fractions of newly synthesized PreProPTH, but not of ProPTH, to limited proteolysis indicates that the two precursors are located in different subcellular compartments and suggests that PreProPTH is converted to ProPTH before its entry into the intracisternal space of the endoplasmic reticulum. Alternatively, the PreProPTH identified in parathyroid gland slices may represent polypeptide chains synthesized in the cell sol on polyribosomes that are not attached to endoplasmic reticulum but are adsorbed nonspecifically to the particulate fraction of the cell during the process of tissue homogenization.

Animals↗
PubMed1979

Immunocytochemical localization of parathyroid hormone in bovine parathyroid glands and human parathyroid adenomas.

Light and electron microscopic localization of parathyroid hormone (PTH) in human and bovine parathyroid tissue has been achieved using an indirect peroxidase labeled antibody method. Granular deposition of the reaction product was found throughout the chief cell cytoplasm. There was no nuclear staining. At the ultrastructural level, parathyroid hormone localized by this method appeared to be largely confined to the secretory granules in the cytoplasm of cells. Mitochondria and nuclei were free of reaction product. Aggregated sacs of granular endoplasmic reticulum were minimally reactive, and Golgi apparatuses did not show reaction product.

Adenoma↗
PubMed1978

Pre-proparathyroid hormone; amino acid sequence, chemical synthesis, and some biological studies of the precursor region.

The precursor of bovine proparathyroid hormone was synthesized by translation of parathyroid mRNA in a wheat-germ cell-free system. The amino acid sequence of the NH2-terminal extension (the pre sequence) was determined by repetitive Edman degradation of the polypeptide labeled with radioactive amino acids (radiosequencing). The pre sequence of pre-proparathyroid hormone is (formula: see text) which is followed by the sequence of proparathyroid hormone. It is significant that 20 of the 25 amino acids in the sequence are hydrophobic. This high hydrophobicity is consistent with the proposed role of the pre sequence as a membrane-penetrating peptide. The precursor-specific sequence of 31 amino acids was snythesized chemically by the solid-phase technique. This synthetic peptide was shown to bind to the microsomal fraction of homogenates prepared from extracts of parathyroid glands, a finding consistent with the proposed role of the precursor peptide in the attachment of the nascent chain--mRNA--ribosome complex to the endoplasmic reticulum.

Amino Acid Sequence↗