The diabetogenic antibiotic streptozotocin modifies the tryptic digest pattern for peptides of the enzyme O-GlcNAc-selective N-acetyl-β-d-glucosaminidase that contain amino acid residues essential for enzymatic activity

2006 ◽  
Vol 72 (6) ◽  
pp. 710-718 ◽  
Author(s):  
Thomas N. Lee ◽  
William E. Alborn ◽  
Michael D. Knierman ◽  
Robert J. Konrad
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Residues ◽  
Tryptic Digest

scholarly journals Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes

2010 ◽  
Vol 432 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Emily R. Slepkov ◽  
Alan Pavinski Bitar ◽  
Hélène Marquis
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Phospholipase C ◽  
Bacterial Pathogen ◽  
Amino Acid Residues ◽  
C Terminus ◽  
N Terminus ◽  
Vacuolar Acidification

The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.

scholarly journals Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 113-120 ◽  
Author(s):  
Y Fujimura ◽  
Y Usami ◽  
K Titani ◽  
K Niinomi ◽  
K Nishio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Von Willebrand Factor ◽  
Amino Acid Analysis ◽  
Disulfide Bonds ◽  
Platelet Glycoprotein ◽  
Amino Acid Residues ◽  
Tryptic Digest ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

Hog pancreatic α-amylase peptides from tryptic digest of isozyme AII

1980 ◽  
Vol 45 (9) ◽  
pp. 2572-2582 ◽  
Author(s):  
Bedřich Meloun ◽  
Ivan Kluh ◽  
Ladislav Morávek
Keyword(s):  
Amino Acid ◽  
Paper Chromatography ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
Tryptic Digest ◽  
Methionine Residues

Peptides isolated from the tryptic digest of the S-sulfo derivative of isozyme AII by gel filtration on Sephadex columns, paper chromatography and electrophoresis were sequenced by the phenylisothiocyanate method. The sequential regions determined account for 319 nonoverlapping amino acid residues in 46 peptides. Sequences around methionine residues and problems of isolation of amylase isozymes are discussed.

scholarly journals Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 113-120
Author(s):  
Y Fujimura ◽  
Y Usami ◽  
K Titani ◽  
K Niinomi ◽  
K Nishio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Von Willebrand Factor ◽  
Amino Acid Analysis ◽  
Disulfide Bonds ◽  
Platelet Glycoprotein ◽  
Amino Acid Residues ◽  
Tryptic Digest ◽  
Von Willebrand ◽  
Willebrand Factor

Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

scholarly journals The Dual α-Amidation System in Scorpion Venom Glands

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 425 ◽  
Author(s):  
Gustavo Delgado-Prudencio ◽  
Lourival D. Possani ◽  
Baltazar Becerril ◽  
Ernesto Ortiz
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Biological Function ◽  
Scorpion Venom ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Proprotein Convertases ◽  
Functional Potential ◽  
Recognition Sites ◽  
Venom Glands

Many peptides in scorpion venoms are amidated at their C-termini. This post-translational modification is paramount for the correct biological function of ion channel toxins and antimicrobial peptides, among others. The discovery of canonical amidation sequences in transcriptome-derived scorpion proproteins suggests that a conserved enzymatic α-amidation system must be responsible for this modification of scorpion peptides. A transcriptomic approach was employed to identify sequences putatively encoding enzymes of the α-amidation pathway. A dual enzymatic α-amidation system was found, consisting of the membrane-anchored, bifunctional, peptidylglycine α-amidating monooxygenase (PAM) and its paralogs, soluble monofunctional peptidylglycine α-hydroxylating monooxygenase (PHMm) and peptidyl-α-hydroxyglycine α-amidating lyase (PALm). Independent genes encode these three enzymes. Amino acid residues responsible for ion coordination and enzymatic activity are conserved in these sequences, suggesting that the enzymes are functional. Potential endoproteolytic recognition sites for proprotein convertases in the PAM sequence indicate that PAM-derived soluble isoforms may also be expressed. Sequences potentially encoding proprotein convertases (PC1 and PC2), carboxypeptidase E (CPE), and other enzymes of the α-amidation pathway, were also found, confirming the presence of this pathway in scorpions.

Involvement of some amino acid residues in the enzymatic activity of solubilized cerebral fucosyltransferase

1984 ◽  
Vol 16 (7) ◽  
pp. 829-832 ◽  
Author(s):  
Pierre Broquet ◽  
Mireille Serres-Guillaumond ◽  
Pierre Louisot
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Residues

scholarly journals Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Zhi-Zhong Song ◽  
Bin Peng ◽  
Zi-Xia Gu ◽  
Mei-Ling Tang ◽  
Bei Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Residue ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Peach Fruit ◽  
Alcohol Acyltransferase

AbstractThe aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

scholarly journals Identification of an Antigenic Epitope inHelicobacter pylori Urease That Induces Neutralizing Antibody Production

2001 ◽  
Vol 69 (11) ◽  
pp. 6597-6603 ◽  
Author(s):  
Kaoru Hirota ◽  
Kumiko Nagata ◽  
Yoshihiko Norose ◽  
Seiji Futagami ◽  
Yohko Nakagawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Neutralizing Antibody ◽  
Antigenic Peptides ◽  
Amino Acid Residues ◽  
Antigenic Epitope ◽  
Amino Acid Deletion ◽  
Entire Sequence ◽  
H Pylori ◽  
Insight Into

ABSTRACT We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pyloriinfection.

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