A serum protease cleaves proANF into a 14-kilodalton peptide and ANF

1987 ◽  
Vol 252 (1) ◽  
pp. E147-E151
Author(s):  
K. D. Bloch ◽  
J. B. Zisfein ◽  
M. N. Margolies ◽  
C. J. Homcy ◽  
J. G. Seidman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Culture Medium ◽  
Rat Plasma ◽  
Biologically Active ◽  
Amino Terminal ◽  
Isolation And Characterization ◽  
Amino Acid Precursor ◽  
Acid Precursor

Proatrial natriuretic factor (proANF), the 126-amino acid precursor of ANF, is the major storage form in mammalian atria. In contrast, two ANF peptides containing the 28- and 24-carboxyterminal residues of proANF have been isolated from rat plasma. Whether the cleavage of proANF in vivo to these ANF peptides occurs during or after its release into the circulation has not been determined. The latter possibility was suggested by our previous study where, by using a cultured rat cardiocyte preparation, we demonstrated that proANF is secreted intact into the culture medium. We now report that serum, but not plasma, contains a protease that specifically cleaves the 17-kdalton proANF to a 14-kdalton amino-terminal peptide and the carboxyterminal 3-kdalton circulating forms of ANF. The role of this proANF-cleaving enzyme in the generation of the biologically active ANF peptides remains to be defined. Its isolation and characterization should provide insights into its site of production and whether in vivo it is involved in the processing of circulating proANF.

scholarly journals Roles of Prenyl Protein Proteases in Maturation of Saccharomyces cerevisiae a-Factor

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 95-101
Author(s):  
Victor L Boyartchuk ◽  
Jasper Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Direct Role ◽  
Amino Acid Precursor ◽  
Carboxyl Terminal ◽  
Secreted Peptides ◽  
Pro Region ◽  
Acid Precursor

Abstract In eukaryotes small secreted peptides are often proteolytically cleaved from larger precursors. In Saccharomyces cerevisiae multiple proteolytic processing steps are required for production of mature 12-amino-acid a-factor from its 36-amino-acid precursor. This study provides additional genetic data supporting a direct role for Afc1p in cleavage of the carboxyl-terminal tripeptide from the CAAX motif of the prenylated a-factor precursor. In addition, Afc1p had a second role in a-factor processing that was independent of, and in addition to, its role in the carboxyl-terminal processing in vivo. Using ubiquitin-a-factor fusions we confirmed that the pro-region of the a-factor precursor was not required for production of the mature pheromone. However, the pro-region of the a-factor precursor contributed quantitatively to a-factor production.

scholarly journals A hidden catalysis: metal-, and organocatalyst-free one-pot assembly of chiral aza-tricyclic molecules

2021 ◽  
Author(s):  
Dung Do
Keyword(s):  
Amino Acid ◽  
Michael Reaction ◽  
Chemical Change ◽  
Building Blocks ◽  
One Pot ◽  
Complex Molecules ◽  
Enamine Catalysis ◽  
Amino Acid Precursor ◽  
Acid Precursor

<p></p><p> Development of a rapid synthesis of complex molecules from simple building blocks under a metal-and organocatalyst-free condition is both conceptually and chemically challenging. Here, we developed a hidden catalysis that allow the straightforward assembly of enantiopure aza-tricyclic molecules containing six contiguous stereocenters from <a>aminophenols, α,β-unsaturated aldehydes </a>and α-amino acids. <a>Without using a metal or an organocatalyst, our approach relies on a temporary formation of a spiroimidazolidinone intermediate and its participation in a sequential aza-Michael/Michael reaction as both a substrate and a catalyst</a> under an iminium/enamine catalysis. The formation of the putative iminium intermediate was supported by spectroscopic data and its interruptive reduction derivative was isolated and fully characterized. Whereas a conventional catalyst is always present and does not undergo a permanent chemical change in a classic catalysis, the spiroimidazolidinone intermediate is conceptualized as a sub-catalyst as it is only temporary produced from precursors and catalyzes its own consumption. This unique substrate-catalyst (sub-catalyst) dual role of the spiroimidazolidinone induces a substantial steric discrimination in the transition state and an excellent overall diastereoselectivity (>20:1 dr). It allows the use of an amino acid precursor as the sole chirality genesis and avoids the use of transition metals or organocatalysts. An enantiomer of an aza-tricyclic imidazolidinone can be prepared from a commercially available amino acid precursor. The aqueous-based reaction is practical and scalable for multi-gram synthesis. The success of implementing this sub-catalysis concept in the synthesis will pave the way for many efficient chiral catalyst-free preparations of chiral complex molecules.<br></p><br><p></p>

scholarly journals D-Serine Production, Degradation, and Transport in ALS: Critical Role of Methodology

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
John P. Crow ◽  
John C. Marecki ◽  
Misty Thompson
Keyword(s):  
Amino Acid ◽  
Intracellular Transport ◽  
Critical Role ◽  
Receptor Activation ◽  
Biologically Active ◽  
Receptor Affinity ◽  
Per Se ◽  
The Brain

In mammalian systems, D-serine is perhaps the most biologically active D-amino acid described to date. D-serine is a coagonist at the NMDA-receptor, and receptor activation is dependent on D-serine binding. Because D-serine binding dramatically increases receptor affinity for glutamate, it can produce excitotoxicity without any change in glutamateper se. D-serine is twofold higher in the spinal cords of mSOD1 (G93A) ALS mice, and the deletion of serine racemase (SR), the enzyme that produces D-serine, results in an earlier onset of symptoms, but with a much slower rate of disease progression. Localization studies within the brain suggest that mSOD1 and subsequent glial activation could contribute to the alterations in SR and D-serine seen in ALS. By also degrading both D-serine and L-serine, SR appears to be a prime bidirectional regulator of free serine levelsin vivo. Therefore, accurate and reproducible measurements of D-serine are critical to understanding its regulation by SR. Several methods for measuring D-serine have been employed, and significant issues related to validation and standardization remain unresolved. Further insights into the intracellular transport and tissue-specific compartmentalization of D-serine within the CNS will aid in the understanding of the role of D-serine in the pathogenesis of ALS.

The Pediocin AcH Precursor Is Biologically Active

1999 ◽  
Vol 65 (6) ◽  
pp. 2281-2286 ◽  
Author(s):  
Bibek Ray ◽  
Robin Schamber ◽  
Kurt W. Miller
Keyword(s):  
Amino Acid ◽  
Membrane Damage ◽  
Biologically Active ◽  
Leader Peptide ◽  
Chimeric Proteins ◽  
Cell Protection ◽  
E Coli ◽  
Amino Acid Precursor ◽  
Insight Into ◽  
Acid Precursor

ABSTRACT The properties of the pediocin AcH precursor, prepediocin AcH, have been studied to gain insight into how producer cells may protect themselves from the activity of intracellular prebacteriocins. The native 62-amino-acid precursor and the 44-amino-acid mature species were expressed in Escherichia coli host strains that lack the leader peptide processing enzyme, PapD. Both forms inhibited the growth of the test bacterium Listeria innocua Lin11, indicating that the native precursor is biologically active. The two species also were synthesized in the context of maltose-binding protein chimeric proteins to facilitate the measurement of their relative specific activities. The chimeric form of the precursor was ∼80% as active as the chimeric mature species. Of relevance to cell protection and pediocin AcH production, it was determined that the precursor is strongly susceptible to inactivation by reducing agents and to degradation by chymotrypsin and endogenous E. coliproteases. Taken together, the results indicate that the activity of prepediocin AcH may have to be controlled prior to secretion to prevent toxicity to the host. Perhaps producer cells avoid membrane damage by maintaining the precursor in a reduced inactive state or by degrading molecules whose secretion is delayed.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS

1972 ◽  
Vol 54 (2) ◽  
pp. 279-294 ◽  
Author(s):  
David C. Shephard ◽  
Wendy B. Levin
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Technical Problems ◽  
Hydrolyzed Protein ◽  
Protein Amino Acids ◽  
Amino Acid Labeling ◽  
Labeling Pattern

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with 14CO2 for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of 14CO2 into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with 14CO2 in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.

scholarly journals A synthetic glucagon-like peptide-1 analog with improved plasma stability

1998 ◽  
Vol 159 (1) ◽  
pp. 93-102 ◽  
Author(s):  
U Ritzel ◽  
U Leonhardt ◽  
M Ottleben ◽  
A Ruhmann ◽  
K Eckart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasma Insulin ◽  
Rat Plasma ◽  
Plasma Stability ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

scholarly journals Antagonists of the system L neutral amino acid transporter (LAT) promote endothelial adhesivity of human red blood cells

2017 ◽  
Vol 117 (07) ◽  
pp. 1402-1411 ◽  
Author(s):  
Laura Beth Mann Dosier ◽  
Vikram J. Premkumar ◽  
Hongmei Zhu ◽  
Izzet Akosman ◽  
Michael F. Wempe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Neutral Amino Acid Transporter ◽  
System L

SummaryThe system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.

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