The Kinetics of Carboxypeptidase B Activity

Wolff, Edith C.; Schirmer, Eva; Folk, J.E.

doi:10.1016/s0021-9258(18)50126-5

Cited by 111 publications

(16 citation statements)

References 17 publications

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“…The reciprocal of this value corresponds to a maximal dissociation constant of approximately 2.5 × 10 -18 M for the enzyme−substrate complex in the transition state. This K tx value is slightly lower than those that have been estimated for adenosine deaminase and cytidine deaminase, which also catalyze the hydrolysis of C−N bonds. − , Table shows that an endopeptidase (the angiotensin-converting enzyme) and also a dipeptidase from ascites tumor cells are somewhat less proficient in enhancing the rates of their respective reactions…”

Section: Discussionmentioning

confidence: 72%

Rates of Uncatalyzed Peptide Bond Hydrolysis in Neutral Solution and the Transition State Affinities of Proteases

1996

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To assess the relative proficiencies of enzymes that catalyze the hydrolysis of internal and C-terminal peptide bonds, the rates of the corresponding nonenzymatic reactions were examined at elevated temperatures in sealed quartz tubes, yielding linear Arrhenius plots. The results indicate that in neutral solution at 25 °C, peptide bonds are hydrolyzed with half-times of approximately 500 years for the C-terminal bond of acetylglycylglycine, 600 years for the internal peptide bond of acetylglycylglycine N-methylamide, and 350 years for the dipeptide glycylglycine. These reactions, insensitive to changing pH or ionic strength, appear to represent uncatalyzed attack by water on the peptide bond. Comparison of rate constants indicates very strong binding of the altered substrate in the transition states for the corresponding enzyme reactions, K tx attaining a value of less than 10-17 M in carboxypeptidase B. The half-life of the N-terminal peptide bond in glycylglycine N-methylamide, whose hydrolysis might have provided a reference for assessing the catalytic proficiency of an aminopeptidase, could not be determined because this compound undergoes relatively rapid intramolecular displacement to form diketopiperazine (t 1/2 ∼ 35 days at pH 7 and 37 °C). The speed of this latter process suggests an evolutionary rationale for posttranslational N-acetylation of proteins in higher organisms, as a protection against rapid degradation.

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Section: Discussionmentioning

confidence: 72%

Rates of Uncatalyzed Peptide Bond Hydrolysis in Neutral Solution and the Transition State Affinities of Proteases

1996

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“…Amylase activity in tissue sonicates was detected by a modified Bernfield (1955) assay', measuring maltose equivalent release from starch in 0 .05 M histidine Cl at pH 6 .5. The proteolytic enzymes were assayed by micromodification' of the spectrophotometric methods for chymotrypsin (Hummel, 1959), carboxypeptidase A (Folk and Schirmer, 1963), and carboxypeptidase B (Wolff et al ., 1962) . Prior to assays of proteolytic enzymes, the tissue sonicate was pretreated with crystallized trypsin (Worthington) .…”

Section: Methodsmentioning

confidence: 99%

The Development of the Dorsal and Ventral Mammalian Pancreas in Vivo and in Vitro

Spooner

Walther

Rutter

1970

The Journal of Cell Biology

113

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The origin, morphogenesis, and biochemical differentiation of the dorsal and ventral pancreas of the rat embryo have been investigated in order to ascertain the similarities and dissimilarities between the two lobes. We have utilized a culture system in which the primitive gut gives rise to a number of differentiated organs, including the dorsal and ventral pancreas . The two pancreases do not undergo fusion in these cultures, thus allowing independent analyses of the two lobes for comparison with in vivo results. The dorsal pancreas first appeared at the 23-25 somite stage while the ventral pancreas appeared approximately 12 hr later at the 29-30 somite stage . Guts from embryos as young as 12 somites were capable of developing both pancreases in vitro. In spite of the 12 hr difference between the times of their appearance, the dorsal and ventral pancreases exhibited identical patterns of morphological and biochemical differentiation . The two lobes contained the same exocrine enzymes and hormones, at similar levels, differing only in their glucagon content, the dorsal pancreas possessing a fivefold higher glucagon specific activity . The implications of these results are discussed .

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“…However, the liberation of 99m Tc-IPG-G was not observed when 99m Tc-PG-GfK(Boc) was incubated under same conditions. The formation of 99m Tc-IPG-G from 99m Tc-IPG-GFK(Boc) was almost completely inhibited by phosphoramidon (an inhibitor of NEP 29 ), partially inhibited by MGTA (an inhibitor of metalloenzymes 30 ) and captopril (an inhibitor of angiotensin converting enzyme 31 ). Cilastatin (an inhibitor of dipeptidyl peptidase 32 ) failed to inhibit the liberation of 99m Tc-IPG-G from 99m Tc-IPG-GFK(Boc) (Figure 3).…”

Section: Enzyme Recognition Of the Cleavable Linkages In 99mmentioning

confidence: 99%

Preferential Cleavage of a Tripeptide Linkage by Enzymes on Renal Brush Border Membrane To Reduce Renal Radioactivity Levels of Radiolabeled Antibody Fragments

et al. 2018

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The obstructive renal radioactivity after injection of antibody fragments/constructs labeled with metallic radionuclides would be improved by liberating a radiometal chelate of urinary excretion from the antibody molecules by enzymes on the renal brush border membrane (BBM). A tripeptide GFK sequence was newly evaluated as an enzyme-cleavable linkage and conjugated to a Tc chelate of an isonicotinic acid derivative of 2-picolylglycine (Tc-IPG). Tc-IPG-glycine was liberated fromTc-IPG-GFK by the enzymes, while Tc-IPG-GK (where the tripeptide GFK was substituted with a dipeptide GK) did not. When injected into mice,Tc-IPG-GFK-conjugated Fab exhibited lower renal radioactivity levels than directly radioiodinated Fab shortly after injection without reducing the tumor radioactivity levels, due to a release and excretion of Tc-IPG-glycine by enzymes present on the renal BBM. These findings would provide insights to develop antibody fragments/constructs labeled with metallic radionuclides of the clinical relevance for improved renal radioactivity levels.

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The Kinetics of Carboxypeptidase B Activity

Cited by 111 publications

References 17 publications

Rates of Uncatalyzed Peptide Bond Hydrolysis in Neutral Solution and the Transition State Affinities of Proteases

Rates of Uncatalyzed Peptide Bond Hydrolysis in Neutral Solution and the Transition State Affinities of Proteases

The Development of the Dorsal and Ventral Mammalian Pancreas in Vivo and in Vitro

Preferential Cleavage of a Tripeptide Linkage by Enzymes on Renal Brush Border Membrane To Reduce Renal Radioactivity Levels of Radiolabeled Antibody Fragments

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