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ProSAAS-derived Peptides

ProSAAS is a newly discovered protein with a neuroendocrine distribution generally similar to that of prohormone convertase 1 (PC1), a peptide processing endopeptidase. ProSAAS mRNA is broadly distributed among neurons in brain and other neuroendocrine tissues (pituitary, adrenal, pancreas).

Overexpression of ProSAAS in the AtT-20 cells substantially reduces the rate of processing of POMC  (endogenous prohormone proopiomelanocortin) [1]. In wild type mouse brain and pituitary, the majority of ProSAAS is processed into smaller peptides including Little SAAS, PEN and big LEN. [2] A few obesity related propeptides have been processed by PC1 and obesity has been observed to be associated with mutations in the human PC1 gene[3].   Does proSAAS-derived peptides play  a role in the development of obesity?

1. Fricker, L.D., et al. J Neurosci 20,639-648(2000)

2. Mazhavia N., et al. ProSAAS processing in mouse brain and pituitary. JBC Nov. 27, 2000

3. Jackson, R.S., et al. Nat Gene 16,303-306(1997)

 

Schematic representation of the PC1-specific inhibitor proSAAS. The presence of two PC-like cleavage sites in proSAAS and its site of interaction with the catalytic domain of PC1 are illustrated. The selected proSAAS-(235-246) peptide used in the current study is also indicated. hproSAAS, human proSAAS. Basak A, et al. J Biol Chem. 2001 Aug 31;276(35):32720-8
Dixon plots for inhibition of mPC1 by proSAAS-(235-246) (A) and proSAAS-(235-244) (B). The graphical plots were obtained with data using mPC1 (5 l) and pERTKR-MCA as substrate at concentrations 12.5 (S1), 25 (S2), 50 (S3), and 100 M (S4). The inhibition study was conducted with a 15-min preincubation between the enzyme and the inhibitor, following which the substrate pERTKT-MCA was added. The fluorescence readings were measured after a 6-h reaction at 37 C (see "Experimental Procedures"). hproSAAS, human proSAAS. Basak A, et al. J Biol Chem. 2001 Aug 31;276(35):32720-8
Effect of synthetic proSAAS C-terminal peptides on the activity of purified PC1. The peptides were tested with purified PC1 (63 ng) and 100 M substrate, as described under "Materials and Methods." The data shown were obtained after 90 min of incubation at 25 C. The experiment was performed three times with less than 10% variation. Qian Y, et al. J Biol Chem. 2000 Aug 4;275(31):23596-601
Time-dependent inhibition of mPC1 activity by proSAAS-(235-244) (192 nM). mPC1 (5 l, containing 3.9 nmol of AMC released/h) was preincubated separately with 192 nM of proSAAS-(235-244) for 0, 5, 15, and 30 min. Following each preincubation period, pERTKR-MCA (100 M) was added, and the progress curves were followed by fluorescence for 30 min. The enzyme inhibition was determined by the initial slope of various progress curves. Basak A, et al. J Biol Chem. 2001 Aug 31;276(35):32720-8
Progress curves over a period of 1,000 s showing inhibition of PC1, furin, PACE4, and PC7 activity in the presence of varying concentrations of proSAAS-(235-244). The enzyme activity was measured against the fluorogenic substrate pERTKR-MCA (100 M) in the presence of increasing concentrations of proSAAS-(235-244) (as indicated in the right side of each panel). The release of fluorescence was followed over a period of 10 min. The inset in the PC7 panel is the progress curve monitored over a period of 1 h. Basak A, et al. J Biol Chem. 2001 Aug 31;276(35):32720-8

 

Northern blot analysis of proSAAS mRNA in human tissues. Northern blots containing poly(A) RNA (Clontech) were probed with 32P-labeled human proSAAS cDNA, as described in Materials and Methods, and were exposed to x-ray film for 2 hr at 80C. Fricker LD, et al. J Neurosci. 2000 Jan 15;20(2):639-48
 
 

 

Deletion mutational analysis of the PC1 inhibitory region of proSAAS. The proSAAS-derived peptides previously identified in Cpefat/Cpefat mouse tissues and in AtT-20 cells are indicated by shading; the names of several of these peptides are indicated on the top line and refer to internal amino acid sequences (KEP, SAAS, PEN, and LEN). Single (R) and dibasic (RR or KR) processing sites are indicated. Fusion constructs consisting of GST with C-terminal extensions of the indicated portions of rat proSAAS were created in the pGEX-2T vector (Amersham Pharmacia Biotech) as described under "Materials and Methods." Protein was expressed in bacteria and purified on glutathione-agarose. The amount of protein was quantitated from the absorption at 280 nm, using the calculated extinction coefficient for each construct. The construct 231-246 was created as both a GST fusion protein and as a synthetic peptide, and the constructs 221-242, 245-260, 245-254, 34-59, 34-40, and 42-59 were created only as synthetic peptides. The PC1 assay was performed with 5 M substrate, using media from PC1-expressing baculovirus as described under "Materials and Methods." All constructs were tested at 1 M in at least two separate experiments, with similar results. Yes indicates greater than 50% inhibition, whereas no indicates less than 10% inhibition; none of the constructs showedpartial inhibition. Qian Y, et al. J Biol Chem. 2000 Aug 4;275(31):23596-601

Amino acid identity among human, rat, and mouse proSAAS. Asterisks below the sequence denote residues conserved in all three species. Open arrowhead, Signal peptide cleavage site. Double arrows, Paired basic cleavage sites (RR, KR) that are used in the Cpefat/Cpefat mouse. Single arrows, KxxR cleavage site used in the Cpefat/Cpefat mouse. Half arrows, Additional predicted cleavage sites. The sequences of the peptides that correspond to those found in the Cpefat/Cpefat mice (but without the C-terminal basic residues that would be removed by active CPE) are indicated by lines. Fricker LD, et al. J Neurosci. 2000 Jan 15;20(2):639-48

%SAAS%


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