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Finn Wold - One of the best experts on this subject based on the ideXlab platform.
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studies on the specificity of acetylaminoacyl Peptide Hydrolase
Protein Science, 1994Co-Authors: Charles W Sokolik, T C Liang, Finn WoldAbstract:In a continuing attempt to explore the types of specificity determinants that may affect protein-protein (Peptide) interactions, a number of short (2-5 residues) acetylated Peptides have been compared as substrates for the enzyme acetylaminoacyl-Peptide Hydrolase (EC 3.4.19.1). The reference substrate was Ac-AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat for the different substrates were determined by standard steady-state kinetics, and the corresponding delta delta GT++ value derived from kcat/Km was used for the comparison, setting delta detal GT++ for Ac-AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1', 2', or 3' (delta delta GT++ values of 2-5 kJ); the negative charge provided by the C-terminus of the substrate also appears to be important, since the amide and O-Me ester derivatives caused a change in delta delta GT++ values of -7 to -8 kJ from the reference Peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar Peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582-589), and the proposed active site model incorporates subsites for both charge-charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make-up of each Peptide and not only the effect of a single residue in a given position.(ABSTRACT TRUNCATED AT 250 WORDS)
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specificity determinants of acylaminoacyl Peptide Hydrolase
Protein Science, 1992Co-Authors: Radha G. Krishna, Finn WoldAbstract:In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-Peptide Hydrolase (EC 3.4.19.1), a number of acetylated Peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the Hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the Peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of Peptides, by extension of the N-terminus with acetylamino acids or acetylPeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal Peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated Peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the Peptides with Pro in the third position were very poor substrates; only the Peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in Hydrolase activity varying with the charge's distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference Peptides without positive charges.(ABSTRACT TRUNCATED AT 250 WORDS)
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Specificity determinants of acylaminoacyl‐Peptide Hydrolase
Protein Science, 1992Co-Authors: Radha G. Krishna, Finn WoldAbstract:In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-Peptide Hydrolase (EC 3.4.19.1), a number of acetylated Peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the Hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the Peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of Peptides, by extension of the N-terminus with acetylamino acids or acetylPeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal Peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated Peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the Peptides with Pro in the third position were very poor substrates; only the Peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in Hydrolase activity varying with the charge's distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference Peptides without positive charges.(ABSTRACT TRUNCATED AT 250 WORDS)
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N-terminal sequence analysis of Nα-acetylated proteins after unblocking with N-acylaminoacyl-Peptide Hydrolase
Analytical Biochemistry, 1991Co-Authors: Radha G. Krishna, Christopher C.q. Chin, Finn WoldAbstract:Abstract The enzyme acylaminoacyl-Peptide Hydrolase represents an attractive reagent for the removal of acetylamino acids from the N-terminus of proteins prior to sequencing. However, the enzyme will not accept intact proteins as substrates, and a blocked protein must consequently be fragmented to generate a relative short blocked Peptide, and all the newly generated amino termini must be blocked with an Hydrolase-resistant reagent before the enzyme can be used to specifically unblock the N-terminus. When a number of N-acetylated proteins (enolase, α-crystallin, ovalbumin, cytochrome c, parvalbumin, superoxide dismutase, and myelin basic protein) were subjected to fragmentation with proteases or cyanogen bromide, treatment with succinic anhydride and exhaustive extraction with ether, and the resulting salt-free, succinylated Peptides were incubated with the Hydrolase, the N-terminal sequence was specifically unblocked. An aliquot of the entire Peptide mixture was applied to the protein sequencer, and a single sequence, corresponding to the known N-terminal sequence starting at residue 2, was obtained. When another aliquot of the same Hydrolase-treated Peptide mixture was treated with the enzyme acylase I, the liberated acetylamino acid was cleaved, and the N-terminal amino acid (residue 1) could be identified by amino acid analysis. The amount of sequence information obtained from different proteins with different fragmentation methods varied considerably; in the case of parvalbumin a sequence of 12 residues was obtained, while for myelin basic protein, only 3 residues could be identified; the other proteins yielded from 5- to 9-residue sequences. Rabbit muscle acetylaminoacyl-Peptide Hydrolase, the enzyme used in the present studies, is itself a blocked protein and was subjected to chymotrypsin digestion, succinylation, and Hydrolase digestion. Subsequent sequencing established the sequence ERQVL-, and acylase treatment gave M as the only free amino acid, demonstrating that the N-terminal sequence of this enzyme is Ac-Met-Glu-Arg-Gln-Val-Leu-, identical to the sequences deduced for other Hydrolases.
Carmela R. Abraham - One of the best experts on this subject based on the ideXlab platform.
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Acyl Peptide Hydrolase Degrades Monomeric and Oligomeric Amyloid-Beta Peptide
Molecular Neurodegeneration, 2009Co-Authors: Rina Yamin, Cheng Zhao, Peter B. O’connor, Ann C. Mckee, Carmela R. AbrahamAbstract:Background: The abnormal accumulation of amyloid-beta Peptide is believed to cause malfunctioning of neurons in the Alzheimer's disease brain. Amyloid-beta exists in different assembly forms in the aging mammalian brain including monomers, oligomers, and aggregates, and in senile plaques, fibrils. Recent findings suggest that soluble amyloid-beta oligomers may represent the primary pathological species in Alzheimer's disease and the most toxic form that impairs synaptic and thus neuronal function. We previously reported the isolation of a novel amyloid-betadegrading enzyme, acyl Peptide Hydrolase, a serine protease that degrades amyloid-beta, and is different in structure and activity from other amyloid-beta-degrading enzymes. Results: Here we report the further characterization of acyl Peptide Hydrolase activity using mass spectrometry. Acyl Peptide Hydrolase cleaves the amyloid-beta Peptide at amino acids 13, 14 and 19. In addition, by real-time PCR we found elevated acyl Peptide Hydrolase expression in brain areas rich in amyloid plaques suggesting that this enzyme's levels are responsive to increases in amyloidbeta levels. Lastly, tissue culture experiments using transfected CHO cells expressing APP751 bearing the V717F mutation indicate that acyl Peptide Hydrolase preferentially degrades dimeric and trimeric forms of amyloid-beta. Conclusion: These data suggest that acyl Peptide Hydrolase is involved in the degradation of oligomeric amyloid-beta, an activity that, if induced, might present a new tool for therapy aimed at reducing neurodegeneration in the Alzheimer's brain.
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Acyl Peptide Hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid‐β Peptide
Journal of Neurochemistry, 2007Co-Authors: Rina Yamin, Sandipan Bagchi, Rick Hildebrant, Andrea Scaloni, Russell L. Widom, Carmela R. AbrahamAbstract:Considerable evidence indicates that the amyloid-β (Aβ) Peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Aβ, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Aβ of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl Peptide Hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Aβ-degrading enzyme, and we show that the degradation of Aβ by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Aβ Peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.
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acyl Peptide Hydrolase a serine proteinase isolated from conditioned medium of neuroblastoma cells degrades the amyloid β Peptide
Journal of Neurochemistry, 2007Co-Authors: Rina Yamin, Sandipan Bagchi, Rick Hildebrant, Andrea Scaloni, Russell L. Widom, Carmela R. AbrahamAbstract:Considerable evidence indicates that the amyloid-β (Aβ) Peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Aβ, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Aβ of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl Peptide Hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Aβ-degrading enzyme, and we show that the degradation of Aβ by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Aβ Peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.
Rina Yamin - One of the best experts on this subject based on the ideXlab platform.
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Acyl Peptide Hydrolase Degrades Monomeric and Oligomeric Amyloid-Beta Peptide
Molecular Neurodegeneration, 2009Co-Authors: Rina Yamin, Cheng Zhao, Peter B. O’connor, Ann C. Mckee, Carmela R. AbrahamAbstract:Background: The abnormal accumulation of amyloid-beta Peptide is believed to cause malfunctioning of neurons in the Alzheimer's disease brain. Amyloid-beta exists in different assembly forms in the aging mammalian brain including monomers, oligomers, and aggregates, and in senile plaques, fibrils. Recent findings suggest that soluble amyloid-beta oligomers may represent the primary pathological species in Alzheimer's disease and the most toxic form that impairs synaptic and thus neuronal function. We previously reported the isolation of a novel amyloid-betadegrading enzyme, acyl Peptide Hydrolase, a serine protease that degrades amyloid-beta, and is different in structure and activity from other amyloid-beta-degrading enzymes. Results: Here we report the further characterization of acyl Peptide Hydrolase activity using mass spectrometry. Acyl Peptide Hydrolase cleaves the amyloid-beta Peptide at amino acids 13, 14 and 19. In addition, by real-time PCR we found elevated acyl Peptide Hydrolase expression in brain areas rich in amyloid plaques suggesting that this enzyme's levels are responsive to increases in amyloidbeta levels. Lastly, tissue culture experiments using transfected CHO cells expressing APP751 bearing the V717F mutation indicate that acyl Peptide Hydrolase preferentially degrades dimeric and trimeric forms of amyloid-beta. Conclusion: These data suggest that acyl Peptide Hydrolase is involved in the degradation of oligomeric amyloid-beta, an activity that, if induced, might present a new tool for therapy aimed at reducing neurodegeneration in the Alzheimer's brain.
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Acyl Peptide Hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid‐β Peptide
Journal of Neurochemistry, 2007Co-Authors: Rina Yamin, Sandipan Bagchi, Rick Hildebrant, Andrea Scaloni, Russell L. Widom, Carmela R. AbrahamAbstract:Considerable evidence indicates that the amyloid-β (Aβ) Peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Aβ, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Aβ of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl Peptide Hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Aβ-degrading enzyme, and we show that the degradation of Aβ by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Aβ Peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.
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acyl Peptide Hydrolase a serine proteinase isolated from conditioned medium of neuroblastoma cells degrades the amyloid β Peptide
Journal of Neurochemistry, 2007Co-Authors: Rina Yamin, Sandipan Bagchi, Rick Hildebrant, Andrea Scaloni, Russell L. Widom, Carmela R. AbrahamAbstract:Considerable evidence indicates that the amyloid-β (Aβ) Peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Aβ, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Aβ of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl Peptide Hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Aβ-degrading enzyme, and we show that the degradation of Aβ by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Aβ Peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.
Radha G. Krishna - One of the best experts on this subject based on the ideXlab platform.
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specificity determinants of acylaminoacyl Peptide Hydrolase
Protein Science, 1992Co-Authors: Radha G. Krishna, Finn WoldAbstract:In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-Peptide Hydrolase (EC 3.4.19.1), a number of acetylated Peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the Hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the Peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of Peptides, by extension of the N-terminus with acetylamino acids or acetylPeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal Peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated Peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the Peptides with Pro in the third position were very poor substrates; only the Peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in Hydrolase activity varying with the charge's distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference Peptides without positive charges.(ABSTRACT TRUNCATED AT 250 WORDS)
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Specificity determinants of acylaminoacyl‐Peptide Hydrolase
Protein Science, 1992Co-Authors: Radha G. Krishna, Finn WoldAbstract:In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-Peptide Hydrolase (EC 3.4.19.1), a number of acetylated Peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the Hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the Peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of Peptides, by extension of the N-terminus with acetylamino acids or acetylPeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal Peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated Peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the Peptides with Pro in the third position were very poor substrates; only the Peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in Hydrolase activity varying with the charge's distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference Peptides without positive charges.(ABSTRACT TRUNCATED AT 250 WORDS)
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Nα-ACYLAMINOACYL-Peptide Hydrolase: SPECIFICITY AND USE TO UNBLOCK N-ACETYLATED PROTEINS
Techniques in Protein Chemistry III, 1992Co-Authors: Radha G. KrishnaAbstract:Publisher Summary This chapter discusses the key aspects of the enzyme specificity and describes a method for fragmenting acetylated proteins, blocking the newly generated N-termini by succinylation before unblocking with Hydrolase and seqencing the N-terminal Peptide. The N-terminal acetyl amino acid can be identified by the treatment of the Hydrolase digest with acylase I and amino acid analysis. Acylaminoacyl-Peptide Hydrolase from rabbit muscle can be used to unblock the N-terminus of N-acetylated proteins for sequencing, after converting Hydrolaseresistant intact proteins to Hydrolase-susceptible Peptides and blocking all the new N-termini by succinylation. After fragmentation of the blocked proteins with proteases or cyanogen bromide and succinylation, the large excess of succinate can be removed by ether extraction and the total Peptide mixture subjected to Hydrolase digestion in the same reaction tube. Sequencing an aliquot of the digest yields the original N-terminal sequence starting with residue 2 as the only unblocked sequence. Treating the remainder of the Hydrolase digest with acylase will cause the hydrolysis of the acetylamino acid and permit the identification of the N-terminal amino acid by amino acid analysis.
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N-terminal sequence analysis of Nα-acetylated proteins after unblocking with N-acylaminoacyl-Peptide Hydrolase
Analytical Biochemistry, 1991Co-Authors: Radha G. Krishna, Christopher C.q. Chin, Finn WoldAbstract:Abstract The enzyme acylaminoacyl-Peptide Hydrolase represents an attractive reagent for the removal of acetylamino acids from the N-terminus of proteins prior to sequencing. However, the enzyme will not accept intact proteins as substrates, and a blocked protein must consequently be fragmented to generate a relative short blocked Peptide, and all the newly generated amino termini must be blocked with an Hydrolase-resistant reagent before the enzyme can be used to specifically unblock the N-terminus. When a number of N-acetylated proteins (enolase, α-crystallin, ovalbumin, cytochrome c, parvalbumin, superoxide dismutase, and myelin basic protein) were subjected to fragmentation with proteases or cyanogen bromide, treatment with succinic anhydride and exhaustive extraction with ether, and the resulting salt-free, succinylated Peptides were incubated with the Hydrolase, the N-terminal sequence was specifically unblocked. An aliquot of the entire Peptide mixture was applied to the protein sequencer, and a single sequence, corresponding to the known N-terminal sequence starting at residue 2, was obtained. When another aliquot of the same Hydrolase-treated Peptide mixture was treated with the enzyme acylase I, the liberated acetylamino acid was cleaved, and the N-terminal amino acid (residue 1) could be identified by amino acid analysis. The amount of sequence information obtained from different proteins with different fragmentation methods varied considerably; in the case of parvalbumin a sequence of 12 residues was obtained, while for myelin basic protein, only 3 residues could be identified; the other proteins yielded from 5- to 9-residue sequences. Rabbit muscle acetylaminoacyl-Peptide Hydrolase, the enzyme used in the present studies, is itself a blocked protein and was subjected to chymotrypsin digestion, succinylation, and Hydrolase digestion. Subsequent sequencing established the sequence ERQVL-, and acylase treatment gave M as the only free amino acid, demonstrating that the N-terminal sequence of this enzyme is Ac-Met-Glu-Arg-Gln-Val-Leu-, identical to the sequences deduced for other Hydrolases.
Erwin E. Sterchi - One of the best experts on this subject based on the ideXlab platform.
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Cloning of the PABA Peptide Hydrolase alpha subunit (PPHα) from human small intestine and its expression in COS-1 cells
FEBS Letters, 1993Co-Authors: Eric Dumermuth, Joyce A. Eldering, Jürgen Grünberg, Weiping Jiang, Erwin E. SterchiAbstract:Abstract PABA Peptide Hydrolase (PPH) from human enterocytes is comprised of two submits, alpha and beta. PPHα is over 70% identical to meprin, a protease isolated from mouse and rat kidney. The enzyme shows a modular organization in that it contains an astacin protease domain, an adhesive domain, an EGF-like domain, and a putative C-terminal membrane spanning domain. Expression of a chimeric meprin-PPHα cDNA in COS-1 cells led to the synthesis of immature, transport-incompetent homodimers. In addition, complex glycosylated forms were detected in the culture medium, suggesting that the enzyme is secreted after proteolytic removal of the membrane anchor.
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expression of the alpha subunit of paba Peptide Hydrolase ec 3 4 24 18 in mdck cells synthesis and secretion of an enzymatically inactive homodimer
FEBS Letters, 1993Co-Authors: Jürgen Grünberg, Eric Dumermuth, Joyce A. Eldering, Erwin E. SterchiAbstract:In this paper, we report the expression of PPHα in the polarized cell line MDCK (Madin Darby canine kidney). In these cells, the enzyme was synthesized m an inactive profonn, which upon treatment with trypsin was activated. The enzyme isolated from cell extracts was core-glycosylated and appeared to be retained in the ER as a homodimer. No PPHα was detectable on the surface of intact cells by immunofluoreseence. However, a complex glycosylated soluble but inactive form was present in the culture medium, suggesting that proteolytic removal of the C-terminal membrane anchoring Peptide leads to the secretion of PPHα.