Biomedical subjects

L A Pon

Publications and source records attributed to L A Pon.

24 records · Page 2Linked to original sources

PubMed1988

Acute stimulation of corpus luteum cells by gonadotrophin or adenosine 3',5'-monophosphate causes accumulation of a phosphoprotein concurrent with acceleration of steroid synthesis.

A protein (ib), which we have detected previously in peptide hormone or cAMP-stimulated corpus luteum cells, is shown to be a phosphoprotein and to be posttranslationally converted into a more acidic phosphoprotein (ia). Phosphorylation is demonstrated by two types of experiments, both using two-dimensional gel electrophoresis. In the first type, gels from [35S]methionine-labeled solubilized cell extracts are compared to gels from such extracts treated with alkaline phosphatase. This in vitro phosphatase treatment converts protein ib quantitatively into protein pb, which is synthesized in vivo only in unstimulated cells. Similarly, ia is converted into pa, the posttranslational product of pb. The second type of experiment demonstrates 32P label incorporation into proteins with the same electrophoretic mobilities as proteins ib and ia. Limited proteolytic digestion of all four proteins from phosphatase-treated and untreated corpus luteum cells shows that the newly detected acidic products, ia and pa, give rise to cleavage patterns similar to those of ib and pb. Further, these patterns resemble those produced by all four such proteins from the adrenal. These findings suggest that in both stimulated corpus luteum and adrenal, a similar protein ib, which accumulates with kinetics and stimulant dose response paralleling those of steroid hormone biosynthesis, is phosphorylated during its synthesis and is degraded by conversion to another phosphoprotein (ia).

Adrenal Glands↗

PubMed1986

Acute ACTH regulation of adrenal corticosteroid biosynthesis. Rapid accumulation of a phosphoprotein.

Two-dimensional gel electrophoresis was used to monitor proteins synthesized in unstimulated control and in adrenocorticotropic hormone (ACTH)- or cAMP-stimulated rat adrenal cells. Four proteins, which have similar proteolytic peptide maps, have been identified. The two found primarily in unstimulated cells are referred to as pb and pa, where pb is the protein with more basic isoelectric point. Similarly, proteins ib and ia were detected only in stimulated cells. The synthesis of pb occurs only in unstimulated cells and that of ib only in stimulated cells. Protein ib accumulates with the same lag time, rate, and stimulant dose response as the increase in steroid hormone synthesis. Pulse-chase studies showed that protein ib is not produced from pb by a post-translational modification. Proteins pb and ib thus seem identical with proteins p and i previously identified in rat adrenal cortex and corpus luteum (Krueger, R.J., and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167, and Pon, L.A., and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6594-6599). The acidic forms, pa and ia, appear after a longer lag time and are produced at a slower rate than the basic forms. Pulse-chase studies showed that the disappearance of the basic form of each protein occurs concurrently with the appearance of the corresponding acidic form. Addition of [32P]orthophosphate to stimulated adrenal cells allowed direct demonstration that proteins ib and ia are phosphorylated. Moreover, alkaline phosphatase treatment of [35S]methionine-labeled, cAMP-stimulated adrenal cells caused a large decrease in the amounts of ib and ia and the appearance of proteins with the same two-dimensional electrophoretic mobilities as pb and pa. These observations suggest that protein ib may mediate stimulation of steroidogenesis, be produced by an ACTH- or cAMP-dependent, cotranslational phosphorylation of protein pb, and be lost by a cycloheximide-insensitive, post-translational conversion to ia.

Adrenal Cortex Hormones↗

PubMed1986

Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones. Rapid induction of a similar protein in both tissues.

Two-dimensional electrophoresis was used to detect a protein (ic) synthesized in rat corpus luteum cells in response to acute stimulation by human chorionic gonadotropin or dibutyryl cyclic AMP. This induced protein ic is isoelectric at pH 6.5 (isoelectric focusing) and has an apparent molecular weight of 28,000 (sodium dodecyl sulfate electrophoresis). The human chorionic gonadotropin or dibutyryl cyclic AMP dose response and time course of synthesis of the protein parallel those of progesterone synthesis in stimulated luteal cells. Additionally, cycloheximide, which inhibits the increase in progesterone formation caused by human chorionic gonadotropin or cAMP, also inhibits the synthesis of ic. Proteolytic polypeptide mapping suggests that ic has a very similar primary structure to another protein (pc), which has the same molecular weight as ic, differs from ic in pI, and is synthesized only in unstimulated cells. These polypeptide maps also demonstrate the close similarity of pc and ic to two proteins p and i, synthesized in control and in adrenocorticotropic hormone-stimulated rat adrenal cortex cells, respectively (Krueger, R. J. and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167). In both adrenal cortex and corpus luteum, binding of a tissue-specific polypeptide hormone acts via cAMP to cause increased steroidogenesis and induction of the synthesis of protein i (ic), with the same time course and hormone dose dependence. Also in both tissues, inhibition of protein synthesis at the level of translation (e.g. by cycloheximide addition) causes inhibition of i (ic) synthesis and of stimulated steroid production. This close correlation between the two different tissues in conditions which cause induction of the synthesis of these proteins suggests that the proteins may be common intermediaries in the control by polypeptide hormones of steroidogenesis in endocrine tissues.

Adipose Tissue↗

PubMed1986

Acute cAMP stimulation in Leydig cells: rapid accumulation of a protein similar to that detected in adrenal cortex and corpus luteum.

Addition of cAMP (as the dibutyryl compound) to a primary culture of mouse Leydig cells caused the accumulation of a protein, i(b), with the same dependence on cAMP concentration as the increase in testosterone synthesis. Stimulation of both protein i(b) and testosterone production were inhibited by cycloheximide. Additionally, cAMP caused repression of synthesis of another protein, p(b), with the same approximate molecular weight (28,000 daltons) as i(b), but more basic isoelectric point. This behavior resembles an event which has been documented in the adrenal cortex (Krueger, R.J., and Orme-Johnson, N.R., J. Biol. Chem., 258, 10159-10167, 1983) and corpus luteum (Pon, L.A., and Orme-Johnson, N.R., J. Biol. Chem., 261, 6594-6599, 1986). The discovery of these proteins in a third steroid-producing cell type and the close correlation between conditions causing increased steroid synthesis and increased i(b) production is further indication that protein i(b) may be an intermediary in peptide hormone or cAMP control of steroid hormone biosynthesis.

Adrenal Cortex↗

PubMed1983

Preparation of semisynthetic insulin analogues from bis(tert-butyloxycarbonyl)-desoctapeptide-insulin phenylhydrazide: importance of the aromatic region B24-B26.

Semisynthetic analogues of insulin were prepared from derivatives of desoctapeptide-(B23-30)-insulin (DOI). A1, B1-(Boc)2-DOI (di-Boc-DOI) was converted to A1, B1-(Boc)2-DOI-B22-phenylhydrazide (di-Boc-DOI-NHNH-C6H5) by the trypsin-catalyzed addition of phenylhydrazine in aqueous organic solvents at pH 6.5 [Canova-Davis, E., & Carpenter, F. H. (1981) Biochemistry 20, 7053-7058]. Treatment of di-Boc-DOI-NHNH-C6H5 with BNPS-skatole produced the phenyldiimide. The latter was coupled with a variety of protected peptides that, after removal of protecting groups, yielded the following compounds whose biological activities were compared to that of insulin in binding, in stimulation of hexose transport (), and in the stimulation of lipogenesis [)), in terms of percent of insulin activity, all in the isolated epididymal fat cell: di-Boc-DOI 0.2, (0.1), [0.2]; di-Boc-DOI-NHNH-C6H5 0.5, (0.2), [0.5]; DOI 0.2, (0.2), [0.1]; DOI-(Gly)B23 0.2, (0.2), [0.1]; DOI-(Gly-Phe)B23-24 6.3, (6.3), [8.0]; DOI-(Gly-Phe-Phe)B23-25 17.0, (25.6), [24.7]; DOI-(Gly-Phe-Phe-Tyr)B23-26 59.0, (50.0), [69.0]. The semisynthetic derivatives represent a stepwise readdition of the aromatic residues near the C terminus of the B chain. A given analogue demonstrated comparable activity in all three biological assays. The results indicate that the stepwise addition of aromatic residues to the B-chain C terminus of DOI produces an increase in insulin-like activity. The biological activity of DOI-(Gly-Phe-Phe-Tyr)B23-26, the derivative in which the aromatic region has been completely reassembled, is the same order of magnitude as that of insulin.

Adipose Tissue↗

PubMedDate not supplied

Protein synthesis requirement for acute ACTH stimulation of adrenal corticosteroidogenesis.

The rapid, cAMP mediated increase produced by ACTH in adrenal corticosteroidogenesis depends also on the rapid synthesis of protein. This obligatory involvement of protein synthesis has been established in several laboratories mainly on the basis of studies demonstrating a parallelism, under a variety of experimental conditions, of the capacity of adrenal cells to synthesize protein with their ability to increase steroid production in response to ACTH or cAMP. More recent correlative studies have disclosed the existence of two proteins, denoted as p and i. Protein i appears only in ACTH or cAMP stimulated cells and with the same time course and ACTH dose response as the increase in corticosteroid synthesis. Protein p, which closely resembles i in molecular weight and primary structure but differs in pI, is synthesized only in unstimulated cells. Neither the function of these two proteins nor the exact nature of the interplay of their synthesis on regulation has been established, although it seems reasonably certain that i is stimulatory rather than p inhibitory. The protein glycosylation inhibitor tunicamycin was found to inhibit the ACTH produced increase in steroidogenesis and also to inhibit the synthesis of protein i under conditions which did not inhibit overall protein synthesis. Therefore it seems probable that i is an N-glycosylated form of protein p and that adrenal cells are unresponsive to acute ACTH action if they are incapable of synthesizing glycosylated proteins specifically.

Adrenal Cortex↗