Carboxypeptidase assay

The potential of the MALDI-MS-based assay scheme for the quantification of low molecular weight products and substrates directly from reaction mixtures has been described by Bungert et al. [8]. The glucose oxidase-based conversion of glucose to gluconolactone and the carboxypeptidase A-mediated cleavage of hippuryl-L-phenylalanine were chosen as model systems (Fig. 8.4). 

The enzymatic colorimetric format is followed by the Penzyme test. This test is a qualitative enzymatic assay for rapid detection of -lactam residues in milk (28-30). The detection principle of the Penzyme test is based on measurement of the degree of inactivation of the enzyme oo-carboxypeptidase is involved in the synthesis of the bacterial cell wall by -lactam antibiotics. These residues bind specifically with the enzyme and inactivate it, thus interfering with bacterial cell wall formation. 

In a recent study, Lin et al. (94a) has found that although the activity of PIR is low it is not zero. In carefully purified material assayed with C>p at pH 6 the Km was about twice that of RNase-A while the turnover number was 0.5% of the native value. This material bound the inhibitors 2 -CMP about 12 times less strongly than RNase-A. When Phe 120 is removed with carboxypeptidase from PIR to give des-(120-124)-RNase, all activity and binding properties are lost. 

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

In the presence of a large excess of Co2+, both native (97) and cobalt (92) carboxypeptidase A show an approximately two-fold activity increase. The kinetics of the enzyme are very complex at moderate or high substrate concentrations and involve both apparent activation and inhibition by substrate (95). Under the standard assay conditions used in connection with the observed cobalt activation, all these complicating factors contribute significantly. The additional Co2+ possibly interferes with these secondary effects rather than being a participant in catalysis. Further experimentation is needed to clarify this detail. 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

B19. Brown, K. S., Kingsbary, W. D., Hall, N. W., Dunn, G. L., and Gilvarg, C., Determination of carboxypeptidase A using A-acetyl-phenylalanyl-3-thiaphenylalamine as substrate Application to a direct semm assay. Anal. Bioch. 161,219—225 (1987). 

The first reported preparation of cross-linked enzyme crystals was by Quiocho and Richards in 1964 [1], They prepared crystals of carboxypeptidase-A and cross-linked them with glutaraldehyde. The material they prepared retained only about 5% of the activity of the soluble enzyme and showed a measurable increase in mechanical stability. The authors quite correctly predicted that cross-linked enzyme crystals, particularly ones of small size where the diffusion problem is not serious, may be useful as reagents which can be removed by sedimentation and filtration. Two years later the same authors reported a more detailed study of the enzymic behavior of CLCs of carboxypeptidase-A [2], In this study they reported that only the lysine residues in the protein were modified by the glutaraldehyde cross-linking. The CLCs were packed in a column for a flow-through assay and maintained activity after many uses over a period of 3 months. 

To examine whether unglycosylated acid carboxypeptidase can secreted into the medium, we assayed medium in the presence or absence of tunicamycin by Western blotting analysis. The results indicate that the acid carboxypeptidase was not secreted into the culture medium when tunicamycin was added (Figure 26 B). This suggests that the decrease of enzymatic activity in culture filtrates of mycelia grown in the presence of tunicamycin is largely owing to the decrease of the concentration of the enzyme. 

FI is not frequently used as a readout for carboxypeptidases because the assay principle cannot be applied easily. In C terminally labeled peptide substrates, the primed site part of the car-boxypeptidase recognition sequence is missing, resulting in high KM values, incompatible with the development of robust protease activity assays. The same limitation of the FI assay principle is observed with endopeptidases in which amino acids on the primed site of the substrate (primarily PT) strongly contribute to the binding energy of the peptide. 

Enzymatic assays for aminopeptidases can also be based on the readouts described below, which are applied primarily for endopeptidases and carboxypeptidases. These readouts use peptide substrates that span from the non-primed to the primed side. 

Ferrer, M. et al. 2003 Miniaturizable homogeneous time-resolved fluorescence assay for carboxypeptidase B activity. Anal. Biochem. 317. 94-98. 

Plasma carboxypeptidase N degrades and therefore inactivates bradykinin. This activity may have a role in the regulation of inflammatory peptides. The HPLC method developed for its assay uses the dipeptide hippuryllysine (Hip-Lys) as the substrate and measures activity by measuring the release of hip-puric acid. 

Carboxypeptidase N (kininase I) cleaves C-terminal lysyl and argininyl residues from peptides. The assay described here is about 1000-fold more sensitive... 

Applicability of the assay was shown for human carboxypeptidase N purified from plasma as well as enzyme in unpurified plasma and conditioned medium from a human hepatoma cell line (Hep G2). 

Figure 8. Tracings of stopped-flow fluorescence assays of the hydrolym of Dns-(Gly)g-ia-Phe, 2.5 X catalyzed by zinc and cadmium carboxypeptidase A, 5 X J0-5M, at pH = 7.5 and 25 C in 0.03M Tris-l.OM NaCl (48). Enzyme tryptophans were excited at 285 nm and their emission was measured by means of a bandpass filter peaking at 360 nm.

Figure 10, Phenyl acetate inhibition of carboxypeptidase A-catalyzed hydrolysis of peptide, Dns-(Gly)g-i.-Phe, 1 X and the ester, Dns-(Gly)3-L.-0Phe, 4 X iO M (57), Enzyme concentrations were 5 X iO" and 4 X IO" M for ester and peptide hydrolysis, respectively (48, 57). Assays were performed in the absence (dashed Une) and presence (solid line) of 1 X phenyl acetate, 0Ac, at 25°C and...

Nonradiometric methods iticlude absorbance, fluorescence. and luminescence spectroscopy. Enzyme assays are a common example. The as.suy is usually run at or below the value of thq substrate, with only about S% of the substrate consumed during the assay, and multiple enzyme turnovers occur during the a.ssay. Sometimes enzyme reactions arc coupled, especially if the target reaction does not produce a product that can be detected directly in the assay. An example is carboxypeptidase. which is coupled to the teduction of NADP to NADPH. giving ri.se to absorbance at 340 nm. 

Fig. 3. Time course of 1,10-phenanthroline inhibition, 1 X 10 M OP resulting in 90% inhibition of carboxypeptidase in 60 minutes. Conditions of assay and per cent activity were calculated as in Fig, 2.

Assay of Enzyme Activity. Activity of this enzyme was determined by the increase in ninhydrin color after hydrolysis of Carbobenzoxy-L-glutamyl-L-tyrosine (Z-Glu-Tyr) as substrate at 30, pH 3.1. One katal of acid carboxypeptidase activity was defined as the amount of enzyme required to liberate 1 mol of C-terminal amino acid per second. 

The enzyme exploited in the Penzyme milk test (Neogen Corporation, Michigan, USA) for P-lactam residues is D,D-carboxypeptidase. The assay is based on the following two properties of d, D-carboxypeptidase ... 

More recently, Kang and Storm 171) have shown that conversion of cobalt(II) carboxypeptidase A to the corresponding cobalt (III) enzyme by hydrogen peroxide oxidation is accompanied by the complete loss of peptidase activity when assayed with CbzGly-L-Phe. Nevertheless, the esterase activity, as measured by the rate of hydrolysis of 0-(N-benzoyl-Gly)-D,L-phenyllactic acid, remains unaffected by this transformation. Cobalt(III) complexes are characterized by extremely slow exchange rates due to the transformation from a d (Coll) to a... 

A carboxypeptidase-transpeptidase, which incorporates free diaminopimelic acid into previously formed cell walls, has been isolated from Bacillus mega-terium Membranes from B. megaterium have been solubilized and then reconstituted in a form capable of synthesizing peptidoglycan. Using this reconstituted system, an assay has been developed for a factor necessary for... 

An interesting biochemical assay has been developed for ochratoxin A, which can be spHt into ochratoxin a and phenylalanine by the enzyme carboxypeptidase A with a loss of fluorescence intensity at 380 nm. It is a sensitive assay and has been adapted for the analysis of ochratoxin A in blood samples and seta. 

The use of Procion-dyed Sephadexes and Blue Dextran 2000 as matrices in substrate-elution chromatography of carboxypeptidase G has been invest gated. Sephadexes containing either fluorescamine or 2,4,6-trinitrophenyl groups have been used as fluorescent or chromophoric substrates, respectively, in assays of dextranase. ... 

Trypsin inhibitors in animal diets are known to cause pancreatic hypertrophy and hyperplasia. We therefore monitored pancreas weight and enzymic contents in all of the above experiments. The pancreata were removed from the chicks at the termination of the experiments, weighed, and assayed for activities of trypsin, chymotrypsin and carboxypeptidases A and B, after activating their zymogens with either trypsin or enterokinase. 

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