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A S Stern

Publications and source records attributed to A S Stern.

At least 55 records · Page 3Linked to original sources
PubMed1986

Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells.

Pure, E. coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.

Animals↗
PubMed1986

Recombinant human interleukin 1 alpha: purification and biological characterization.

Interleukin 1 (IL 1) is a polypeptide hormone produced by activated macrophages that affects many different cell types involved in immune and inflammatory responses. The cloning and expression of a murine IL 1 cDNA in Escherichia coli encoding a polypeptide precursor of 270 amino acids has been reported, and expression of the carboxy-terminal 156 amino acids of this precursor in E. coli yields biologically active IL 1. By using the murine IL 1 cDNA as a probe, we have isolated its human homolog from cDNA generated to lipopolysaccharide-stimulated human leukocyte mRNA. Nucleotide sequence analysis of this cDNA predicts a protein of analysis of this cDNA predicts a protein of 271 amino acids (termed IL 1 alpha) which shows congruent to 61% homology to its murine counterpart but only 27% homology to a recently characterized human IL 1 precursor (IL 1 beta). We have expressed the carboxy-terminal 154 amino acids of IL 1 alpha in E. coli, purified this protein to homogeneity, and have compared it with pure recombinant murine IL 1 in several different IL 1 assays based on murine and human cells. Recombinant IL 1 is capable of stimulating T cell and fibroblast proliferation and inducing fibroblast collagenase and prostaglandin production, thus proving that a single molecule has many of the activities previously ascribed to only partially purified IL 1 preparations. Our results indicate that there exists a family of at least two human IL 1 genes (alpha and beta) whose dissimilar protein products have similar biological activities.

Amino Acid Sequence↗
PubMed1986

Structure-function analysis of murine interleukin 1: biologically active polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of a 270-amino acid precursor.

Murine interleukin 1 (IL-1) is initially synthesized as a 270-amino acid precursor protein. Guided by amino-terminal end sequence analyses of mouse macrophage-derived IL-1, it was shown that expression of the carboxyl-terminal 156 amino acids (i.e., amino acids 115-270) of this precursor in Escherichia coli yields biologically active recombinant IL-1 (rIL-1) protein. To answer questions about precursor processing and the size of the smallest biologically active IL-1 fragment, we have engineered deletions of the rIL-1 (115-270) gene to encode two amino-terminal deletion analogs, rIL-1 (131-270) and rIL-1 (144-270), and a carboxyl-terminal deletion analog, rIL-1 (131-257, 270). The analogs were produced in E. coli, purified to homogeneity, and assayed for biological activity on murine thymocytes, human rheumatoid synovial cells, and human dermal fibroblasts and for their ability to bind to IL-1 receptors on murine EL-4 thymoma cells. The amino-terminal deletion analog rIL-1 (131-270) possessed a specific activity in the murine thymocyte proliferation assay equivalent to that of the 115-270 parent protein and exhibited significant biological activity in stimulating the production of collagenase and prostaglandin E2 by synovial cells and fibroblasts. The more extensive amino-terminal deletion analog rIL-1 (144-270) was inactive in all biological assays and failed to compete in the receptor binding assay. The carboxyl-terminal deletion analog rIL-1 (131-257, 270) competed less efficiently (by a factor of 100) in the receptor binding assay, retained weak biological activity on synovial cells and fibroblasts, and only demonstrated full intrinsic activity in the thymocyte proliferation assay when 100-200 times more protein was assayed. These results suggest that biologically active murine IL-1 polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of the 270-amino acid precursor. Furthermore, it appears that the integrity of the carboxyl terminus of the 270-amino acid precursor is important for activity but that different amino termini can be utilized to generate molecules with equivalent specific activities. This amino-terminal end flexibility supports a processing model for IL-1 maturation that partially explains IL-1 polypeptide heterogeneity.

Amino Acids↗
PubMed1985

New bovine adrenal medullary peptide and its precursor.

We have isolated a previously unknown peptide and its precursor from bovine adrenal medullary chromaffin granules. The peptide sequence is Leu-Pro-Val-Asn-Ser-Pro-Met-Asn-Lys-Gly-Asn-Glu-Val-Met-Lys. The peptide is cleaved from the precursor at a Lys site. The sequence shows no homology to any known protein in the largest sequence data bank available.

Adrenal Medulla↗
PubMed1984

Purification to homogeneity and partial characterization of interleukin 2 from a human T-cell leukemia.

A method utilizing reversed-phase high-performance liquid chromatography has been developed for the purification to homogeneity of interleukin 2 (IL-2) isolated from a human T-cell leukemia. A final purification of 500,000-fold was obtained with a specific activity of pure IL-2 of 10(9) units/mg. The amino acid analysis of natural IL-2 is strikingly similar to the composition deduced from sequence analysis of a cDNA coding for human IL-2. Protein sequence analysis of CNBr-derived peptides yields data consistent with the sequence proposed from cloned cDNA. The availability of homogeneous IL-2 will allow accurate biological studies of its activity free from the contamination of the numerous lymphokine species that are known to be co-produced with IL-2 during the induction procedure.

Amino Acid Sequence↗
PubMed1983

Biosynthesis of the enkephalins and enkephalin-containing polypeptides.

The biochemical characterization of the enkephalins and the enkephalin-containing polypeptides and their biosynthesis has reached an advanced state. From the structural data showing great similarities between human and beef proenkephalin there is strong evidence for physiological roles for some of the extended enkephalins and for at least two of the ECPs. If, like the different hormones contained within proopiocortin, the different ECPs in proenkephalin perform different coordinate functions, their cosecretion with the catecholamines from the adrenal gland suggests that these functions play a role in the response to stress. It appears that proenkephalin represents another multi-hormone precursor, like proopiocortin, that is processed by proteolysis to yield a variety of peptides active in both the endocrine and nervous systems. The research effort must now turn toward resolving the nature of the physiologic functions these peptides perform.

Amino Acid Sequence↗
PubMed1982

Opioid polypeptides in guinea pig pancreas.

Extracts from guinea pig pancreas were found to contain high molecular weight enkephalin-containing polypeptides as well as free [Met]enkephalin and [Leu]enkephalin and the hexa-, hepta-, and octapeptides derived from proenkephalin. Within the limits of sensitivity of our assay (10 fmol/g of tissue), beta-endorphin and beta-lipotropin were undetectable.

Amino Acid Sequence↗
PubMed1982

Adrenal opioid proteins of 8600 and 12,600 daltons: intermediates in proenkephalin processing.

[Met]Enkephalin-containing proteins of 8600 and 12,600 daltons have been isolated from acid extracts of bovine adrenal medulla and purified to homogeneity, and their sequences have been determined by a combination of automated Edman degradation, tryptic mapping, and enzymatic time-course hydrolysis. The 8600-dalton protein contains one copy of the [Met]enkephalin sequence at the COOH terminus and the 12,600-dalton protein contains three copies of [Met]enkephalin, of which two are internal and the third is at the COOH terminus. They possess identical NH2-terminal amino acid sequences, suggesting that the 8600-dalton protein is derived from the 12,600-dalton protein by intracellular proteolytic processing. This is supported by results from tryptic maps of both proteins. Furthermore, chemical analysis of the tryptic peptides obtained from the 12,600-dalton protein indicates that it also contains the amino acid sequence that corresponds to a previously characterized enkephalin-containing polypeptide of 3800 daltons (peptide F) [Jones et al. (1980) Arch. Biochem. Biophys. 204, 392-395]. All three polypeptides appear to be intermediates in posttranslational processing of a still larger polyenkephalin precursor molecule, proenkephalin, and part of a biosynthetic pathway leading to smaller enkephalin-containing polypeptides and free enkephalins.

Adrenal Medulla↗
PubMed1982

An 18,200-dalton adrenal protein that contains four [Met]enkephalin sequences.

An 18,200-dalton adrenal enkephalin-containing polypeptide was purified to homogeneity and shown to contain four internal [Met]enkephalin sequences. Protein sequence analysis and mapping of trypsin-derived peptides demonstrated that the previously described 12,600-dalton and 5,300-dalton adrenal enkephalin-containing polypeptides compose the amino-terminal and carboxyl-terminal sequences, respectively, of this larger protein. They are linked by two basic amino acid residues to form the entire sequence of the 18,200-dalton protein. This protein is the largest enkephalin-containing polypeptide yet isolated from beef adrenal medulla and serves as the precursor for several other previously characterized adrenal enkephalin-containing proteins and peptides.

Adrenal Medulla↗
PubMed1981

Two adrenal opioid polypeptides: proposed intermediates in the processing of proenkephalin.

Two enkephalin-containing polypeptides of 3600 and 4900 daltons have been isolated from extracts of bovine adrenal medulla, purified to homogeneity, and analyzed by a combination of automated Edman degradation and enzymatic time course hydrolysis. The 4900-dalton polypeptide contains two copies of enkephalin, one an internal [Met]enkephalin sequence, the other a [Leu]enkephalin sequence at the carboxyl terminus. Sequence analysis of the 3600-dalton polypeptide has not been completed, but the polypeptide has been shown to contain a single [Met]enkephalin sequence followed by an -Arg-Phe linkage that forms the carboxyl terminus of the molecule. On the basis of these and other findings, we propose that the above enkephalin-containing polypeptides are intermediates in the biosynthesis of the enkephalins and that they are generated by posttranslational processing from a large multivalent enkephalin precursor molecule, proenkephalin. The term "multivalent" is used to indicate a polypeptide with many identical functional sequences.

Adrenal Medulla↗
PubMed1981

A highly potent 3200-dalton adrenal opioid peptide that contains both a [Met]- and [Leu]enkephalin sequence.

A 3200-dalton adrenal enkephalin-containing peptide, designated peptide E, that exhibits high opiate activity in the guinea pig ileum longitudinal muscle preparation was purified, and its structure was determined. It contains an amino-terminal [Met]enkephalin sequence and a [Leu]enkephalin sequence at the carboxyl terminus. Sequence analysis revealed that peptide E arises from a previously characterized 4900-dalton adrenal enkephalin-containing peptide. Peptide E was shown to be 30 times more potent than [Met]enkephalin in the guinea pig ileum assay, which suggests that the adrenal enkephalin-containing peptide may perform a unique biological function in vivo.

Adrenal Medulla↗
PubMed1981

Marked increases in large enkephalin-containing polypeptides in the rat adrenal gland following denervation.

When rat adrenal glands are denervated, large increases in the amounts of enkephalin and enkephalin-containing polypeptides appear. In the normal gland, only trace amounts occur. One of the larger polypeptides (approximately 22,000 daltons) increases rapidly and by 48 hr following denervation, attains 20 times its original level. At this time, the levels of free enkephalins are essentially unchanged. By 96 hr, the 22,000-dalton polypeptide begins to decrease as free enkephalins and intermediate-sized enkephalin-containing polypeptides increase. This series of events is consistent with a precursor (22,000-dalton polypeptide)/product (enkephalin) relationship.

Adrenal Glands↗
PubMed1980

An about 50,000-dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin.

A protein that may be an enkephalin precursor has been identified in extracts of bovine adrenal medulla. This protein (about 50,000 daltons) appears to contain seven copies of [Met]enkephalin and one copy of [Leu]enkephalin. Digestion with trypsin and carboxypeptidase B yields [Met]enkephalin and [Leu]enkephalin in a ratio of almost 7 to 1. The enkephalins were identified by chromatography and by their binding to opiate receptors. Some characteristics of several other adrenal peptides that may serve as intermediates in the biosynthesis of the enkephalins are presented.

Adrenal Medulla↗
PubMed1980

Probable precursors of [Leu]enkephalin and [Met]enkephalin in adrenal medulla: peptides of 3-5 kilodaltons.

Adrenal chromaffin granules contain at least 10 peptides, ranging in size from 3 to 5 kilodaltons, that yield, upon digestion with trypsin, peptides that show specific binding to opiate receptors. All are distinctly different from beta-endorphin. Two of these peptides have been purified to homogeneity and subjected to chemical analysis. One is apparently a [Met]enkephalin precursor containing two copies of the [Met]enkephalin sequence. The other peptide contains both [Leu]enkephalin and [Met]enkephalin sequences and is presumably a common precursor of the two forms of enkephalin.

Adrenal Medulla↗