Regulation Of Pro-Opiomelanocortin (Pomc) Biosynthesis In The Amphibian And Mouse Pituitary Intermediate Lobe
Mac E. Hadley in The Melanotropic Peptides, 1988
Two other proteases, an aminopeptidase B-like enzyme and a carboxypeptidase B-like enzyme, are necessary for the removal of the N- and C-terminal-extended basic residues, respectively, from the cleaved peptides following PCE action. A carboxypeptidase B-like enzyme has been found in secretory vesicles of pituitary and chromaffin cells.53,54 This enzyme has been purified and characterized as a metalloprotease, which is stimulated by Co+ +. The enzyme has recently been cloned and the amino acid sequence derived from the cDNA sequence. An aminopeptidase B-like enzyme has been detected in secretory vesicles of pituitary and shown to be a metalloprotease which is also stimulated by Co+ +. (For reviews of the proteolytic prohormone processing enzymes, see References 55,56). However, it seems unlikely that these exopeptidases will play a key role in rate-limiting the production of α-MSH and β-endorphin.
Affinity Modification in Biochemistry, Biology, and Applied Sciences
Dmitri G. Knorre, Valentin V. Vlassov in Affinity Modification of Biopolymers, 1989
The cases are described when strong inhibition due to coordination of the ligand to the metal ion in the active center takes place. Thus, 2-benzyl-3-mercaptopropanoic acid (SQ 14,603) is a strong inhibitor of carboxypeptidase A, specifically cleaving C-terminal aromatic amino acid residues (see Chapter 1, Section I.A). The dissociation constant of the enzymeinhibitor complex is Ki = 1.1 × 10−8M, whereas for carboxypeptidase B specific to C-terminal positively charged amino acid residues Ki = 1.6 × 10−4 M. At the same time 2-mercaptomethyl-5-guanidinopentanoic acid (SQ 24,798) has a Ki of 4 × 10−10M vs. carboxypeptidase B and a Ki of 1.2 × 10−5M vs. carboxypeptidase A. In this case the inhibitory action is due to coordination to a catalytically essential Zn2+ ion.395
Enzymatic Methods of Protein/Peptide Sequencing from Carboxyterminal End
Ajit S. Bhown in Protein/Peptide Sequence Analysis: Current Methodologies, 1988
The first digestion is performed with carboxypeptidase B in 0.1 M ammonium bicarbonate, pH 8.0, and an aliquot of the mixture is subjected to analysis of amino acids released. The remainder of the mixture is successively incubated with carboxypeptidase A and the aliquot is withdrawn at an appropriate time interval for the determination of amino acids released. In this method, it is possible to clearly determine whether any basic residues released are at the C terminus.
Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry
Published in mAbs, 2019
Florian Füssl, Anne Trappe, Ken Cook, Kai Scheffler, Oliver Fitzgerald, Jonathan Bones
Using our native CVA-MS approach, we were able to obtain very high chromatographic resolution and successful identification of the majority of the 16 charge variants observed. Among them were proteoforms modified by different levels of C-terminal lysine truncation, deamidation, succinimide aspartic acid (Asp) formation, glycation and fragmentation. To investigate whether host cell proteins (HCPs) might be involved in the fragmentation of adalimumab, we performed an HCP analysis employing peptide mapping. Several HCPs were detected, among them the protease Cathepsin L, which could suggest the occurrence of the detected fragments to partly be due to enzymatic proteolysis. Carboxypeptidase B (CpB) digestion and peptide mapping experiments were performed as orthogonal methods to confirm the results obtained on the intact protein level.
Assessment of structural and functional similarity of biosimilar products: Rituximab as a case study
Published in mAbs, 2018
Neh Nupur, Nidhi Chhabra, Rozaleen Dash, Anurag S. Rathore
CEX-HPLC was performed to quantify charge variants on MAbPac SCX-10 RS, BioLC™ (4.6 × 250 mm, 5 μm, Thermo Scientific) column operated at 25°C using Dionex Ultimate 3000 RSLC system (Thermo Scientific) consisting of a quaternary pump with a degasser, an auto sampler with a cooling unit, and a DAD. Prior to injection, the column was saturated with 95% mobile phase A (15 mM sodium phosphate buffer and 0.05% NaN3 at pH 6.2) and 5% mobile phase B (150 mM sodium phosphate buffer and 0.05% NaN3 at pH 6.2). All buffers were filtered with a 0.22 μm cutoff nylon membrane filter (Pall Corporation) and degassed prior to use. 20 μg sample was loaded and differently charged species were separated using a 13 min sigmoidal gradient from 5% – 35% B at a flow rate of 1 ml/min.41 Detection was performed by monitoring UV absorbance at 280 nm. For C terminal lysine cleavage, 15 μL of 1 mg/mL carboxypeptidase B (CpB, Sigma-Aldrich; Cat# C9584) was added to 1 mL of sample and the mixture was incubated at 37°C for 30 min. Chromeleon software (Thermo Scientific) was used for chromatogram integration and estimating acidic, basic and main content through relative peak area percentage values.
Quality comparability assessment of a SARS-CoV-2-neutralizing antibody across transient, mini-pool-derived and single-clone CHO cells
Published in mAbs, 2022
Gangling Xu, Chuanfei Yu, Wenbo Wang, Cexiong Fu, Hongchuan Liu, Yanping Zhu, Yuan Li, Chunyu Liu, Zhihao Fu, Gang Wu, Meng Li, Sha Guo, Xiaojuan Yu, Jialiang Du, Yalan Yang, Maoqin Duan, Yongfei Cui, Hui Feng, Lan Wang
CEX HPLC was performed on an e2695 HPLC system (Waters Corporation) equipped with a UV detector set at 280 nm and a MabPac SCX-10 column (10 μm, 4.0 × 250 mm; Thermo Fisher Scientific). Samples were diluted to 1.0 mg/mL with mobile phase A (20 mM of MES; pH: 6.0) and treated with 250:1 (v/v) (protein:enzyme) of carboxypeptidase B (1 mg/mL) at 37°C for 1 h. Approximately 100 μL of sample was injected onto the column at 60% mobile phase A and 40% mobile phase B (20 mM of MES + 100 mM of NaCl; pH: 6.0). After 3 min, the sample was eluted with a linear gradient of 40–80% mobile phase B within 40 min. The LC flow rate was fixed at 1.0 mL/min and the temperature of the column was 40°C.
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