Alfred Chung

New Substrates for the Radioassay of Angiotensin Converting Enzyme of Endothelial Cells in Culture

To develop means of measuring angiotensin converting enzyme of endothelial cells in culture, we have synthesized benzoyl-Phe-Ala-Pro-OH (I), benzoyl-Pro-Phe-Arg-OH (II) and benzoyl-Gly-His-Leu-OH (III), each bearing a 3H-atom on the para-position of its benzoyl moiety. All three of the acylated tripeptides are substrates for the enzyme. Substrate I exhibits the lowest Km (12.5 micrometer) and yields the most sensitive assay: the enzyme of 10(6) cells can be measured in a 30 min incubation at 37 degrees C. Radiolabelled reaction product is separated from substrate by extraction of acidified reaction mixture with an organic solvent, and the rate of formation of product can be quantified by liquid scintillation counting of the organic phase. Substrate III can also be used to measure angiotensin converting enzyme of cells but requires longer incubations (180--240 min) and high salt concentrations (0.75 M Na2SO4). Substrate II is not specific: it is hydrolyzed by more than one enzyme of endothelial cells.

Localization of angiotensin converting enzyme (kininase II). I. Preparation of antibody-heme-octapeptide conjugates

Antibodies to pig lung angiotensin converting enzyme (kininase II) were conjugated to a heme-octapeptide (8-microperoxidase, 8-MP) derived from cytochrome c. 8-MP, which has only one reactive amine, was coupled to antibody in a two-step procedure using a bifunctional active ester, bis-succinyl succinate. In the first-step, 8-MP-succinyl succinate, a stable compound which can be stored. In a second step, the remaining active ester was used for coupling to reactive amines of the antibody. The conjugate consists of 1.6-2.3 8-MP moieties per antibody. Using these procedures, the formation of complex polymers is avoided. Each molecule of conjugate possesses both immunoreactivity and peroxidatic activity. The conjugate has been used to localize angiotensin converting enzyme along the plasma membrane and associated caveolae of pig aortic endothelial cells in culture.

Slow Tight Binding Inhibitors of Angiotensin Converting Enzyme

We have found that apparent Ki values of some, but not all, carboxyalkyl-dipeptide inhibitors of angiotensin converting enzyme decrease as a function of incubation time. The most potent of the ACE inhibitors tested so far is RAC-X-65 (N-[1(S)-carboxy-3-carboxanilidiopropyl]-L-Ala-L-Pro). When RAC-X-65 is not preincubated with human serum ACE (2.4 X 10(-11) M), the apparent Ki value is 4.4 X 10(-10) M. Preincubation of RAC-X-65 with ACE for 15 min before addition of substrate yields an apparent Ki of 4.1 X 10(-11) M. a 90 min preincubation of the inhibitor with ACE yields an apparent Ki of 1.2 X 10(-11) M, i.e., the reaction of the inhibitor with enzyme is virtually stoichiometric. The enzyme:inhibitor complex is poorly separated by molecular sieve chromatography or by dilution. That such tightly bound complexes are formed in vivo is suggested by the following results: The intravenous ED50 (anesthetized rats) of RAC-X-65 is 9.43 nmol/kg, and the time for half recovery (t1/2) of responsiveness to i.v. angiotensin I, 120 ng/kg, following a cumulative dose of 240 nmol/kg of the inhibitor is 165 min. For comparison, the i.v. ED50 of captopril is 105 nmol/kg, and its t1/2 following a cumulative dose of 240 nmol/kg is 16 min. Implied is the possibility that slow tight binding inhibitors of ACE may be used in a 1 pill per day regimen for the treatment of hypertension.

A radioassay for aminoacylproline hydrolase (aminopeptidase P) activity

A radioassay was developed in which aminoacylproline hydrolase acts on Arg-Pro-Pro-[3H]benzylamide to yield arginine plus Pro-Pro-[3H]benzylamide. By stopping the reaction with base (0.1 M NaOH), the radioactive product is deprotonated to an organophilic form and is separable from the hydrophilic substrate by extraction of the alkaline aqueous solution with an organic solvent. When scintillants are included in the organic solvent, the enzyme:substrate reaction, extraction and quantification of Pro-Pro-[3H]benzylamide can all be conducted using a single liquid scintillation vial. Thus, aminoacylproline hydrolase activity is measured in terms of the rate of release of Pro-Pro-[3H]benzylamide. The substrate is obtainable at greater than 20 Ci/mmol, which enables its use under conditions of first-order enzyme kinetics. Conditions of near-zero order kinetics are readily attained by adding unlabeled substrate (Km 0.7 microM). The substrate is highly reactive (a 1:2000 dilution of guinea pig plasma hydrolyzed greater than 10% of the substrate during a 10 min incubation at 37 degrees C) and specific in that it is not degraded by leucine aminopeptidase, aminopeptidase A or N, dipeptidyl peptidase IV nor prolyl endopeptidase. The assay was used to measure aminoacylproline hydrolase specific activities in tissues of rat and guinea pig. Activity was found in virtually all major tissues of both species, and some guinea pig tissues (e.g. kidney and plasma) were found to be notably rich sources of the enzyme.

Radiolabelled substrates for angiotensin converting enzyme

Six [3H]benzoyl-tripeptides were prepared and tested as substrates for angiotensin converting enzyme. Each was prepared first as its [4-iodo]-benzoyl-analog, and an atom of 3H per molecule was introduced by catalytic dehalogenation in 3H2-gas. Kinetic parameters were measured at 37 degrees C using as buffer 0.05 M Hepes, pH 8.0 containing 0.1 M NaCl and 0.6 M Na2SO4. When the substrates were used at concentrations far below their respective Km values, fractional rates of substrate utilization per unit time for constant enzyme concentration were direct function of respective second order rate constants (Kc/Km). Although absolute values of Kc/Km differed for human enzyme as opposed to rabbit enzyme, relative values of Kc/Km were virtually identical. Similarly, relative rates of substrates utilization during passage through lungs of anesthetized rats were similar to relative values of Kc/Km measured in vitro. Thus, there is now a range of ACE substrates usable, in vitro and in vivo, under conditions of first order enzyme kinetics, conditions under which values of V/Km and Ki can be measured directly.

A simple radioassay for human urinary kallikrein.

We have developed a sensitive, highly selective assay for human urinary kallikrein (HUK) that uses Pro-Phe-Arg-[3H]benzyl-amide as substrate. The substrate was prepared from Pro-Phe-Arg-3-iodo-benzylamide by dehalogenation in 3H2 gas. HUK is measured by its ability to release [3H]benzylamine. The pH optimum is 9.5. Urokinase, plasmin and thrombin do not interfere. The assay can measure as little as 5 ng of HUK in a 15 min incubation at 37 degrees C. Typically, we use 50 microliter of dialyzed urine for HUK assays. Reactions are terminated by adding 0.1 M NaOH, and reaction product is separated from substrate by partitioning with an equal volume of toluene. A sample of the toluene phase is submitted for liquid scintillation counting. As judged by separations obtained on molecular sieve chromatography (Sephacryl), only one urinary enzyme possesses the ability to hydrolyze our substrate. The enzyme MW 45,000, is inhibited by Trasylol but not by soya bean trypsin inhibitor (SBTI). It is reactive with and is inhibited by antibodies prepared against pure HUK.

Inhibition of endothelial‐bound angiotensin converting enzyme, in vivo

1 We determined apparent Ki constants of two inhibitors, captopril and CL242,817, for pulmonary endothelial‐bound angiotensin converting enzyme (ACE) in anaesthetized rabbits. [3H]‐benzoyl‐Phe‐Ala‐Pro was used as the substrate. The apparent kinetic parameters Km and Amax (product of Vmax and microvascular plasma volume) were measured, as was the ratio (Amax/Km) (measured under first order reaction conditions) before and 30s after the i.v. administration of captopril 10 nmol kg−1 or CL242,817, 35 nmol kg−1. 2 Under mixed order reaction conditions, ([S] ≥ Km), apparent Km values increased from 12.2 ± 1.9 μm to 32.9 ± 3.3 μm (P < 0.05) in the captopril‐treated rabbits and from 9.3 ± 2.3 μm to 45.8 ± 9.8 μm (P < 0.05) in the CL242,817‐treated rabbits, indicative of competitive inhibition. However, apparent Amax values decreased from 10.3 ± 2.1 to 4.5 ± 0.8 μmol min−1 (P < 0.05) and 8.9 ± 1.7 to 4.8 ± 0.5 μmol min−1 (P < 0.05), respectively. 3 Under first order reaction conditions ([S] ≪ Km), the Amax/Km ratio decreased from 763 ± 100 to 125 ± 38 ml min−1 (P < 0.05) and 1009 ± 149 to 126 ± 44 ml min−1 (P < 0.05) in the captopril‐ and CL242,817‐treated groups respectively. 4 When the single pass transpulmonary binding of 80 pmol [3H]‐RAC‐X‐65 (an ACE inhibitor) was measured in additional rabbits, a significant (P < 0.05) decrease in RAC‐X‐65 binding was observed 30 s after captopril (80% decrease) or CL242,817 (85% decrease), a result expected for a loss of catalytically active enzyme mass due to tightly bound captopril or CL242,817. 5 These results indicate that, in vivo, both captopril and CL242,817 are competitive, tight binding inhibitors of lung ACE. Furthermore, they suggest means for evaluating the interaction of other potential ACE inhibitors with the pulmonary endothelial membrane‐bound enzyme, in vivo, possibly in phase I clinical trials.

Synthetic Inhibitors of Carboxypeptidase N

Further to explore the functions of carboxypeptidase N (CPN) in vivo, we undertook two studies to find CPN inhibitors of high potency and relatively long duration of action. In each study we examined for inhibition of hydrolysis of [3H]benzoyl-Ala-Arg using pure bovine serum CPN or human serum. In the first such study we synthesized a series of acyl amino acids and acyl di - and tripeptides containing arginine, lysine or both. All proved to be weak inhibitors (Ki = 10(-3) to 10(-4) M). N alpha-carbamoyl-Arg was the strongest: Ki = 3.5 X 10(-5) M. In the second study we prepared S-acyl (thio ester) derivatives of the highly potent CPN inhibitor 2-mercaptomethyl-3-guanidinoethylthiopropionic acid (2-MGP), as certain S-acyl groups markedly increase the duration of captopril, another mercapto-containing compound. Acetyl-, Boc-phenylalanyl-, phenylalanyl-, benzoyl-alanyl-, alanyl-, and Boc-alanyl-2-MGP retained the high potency of 2-MGP in vitro. Although Ala-2-MGP exerted maximum effects in vivo, like those of 2-MGP, the duration of action of Ala-2-MGP was slightly shorter than that of 2-MGP. These results indicate that the mercapto group of 2-MGP can be taken up in some forms of thioester linkage and still remain virtually the full potency of 2-MGP itself. Thus, it appears that a free mercapto function is not essential for the action of 2-MGP.

Preparation of Intrinsically-Labelled Kinins

As part of a program to prepare bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) labelled at high specific radioactivities, we have synthesized three analogs for dehalogenation in tritium gas: [4-Br-Phe5]-bradykinin (BK), [4-Br-Phe8]-BK and [4-Br-Phe5,8]-BK. The analogs were synthesized by the Merrifield solid-phase method and were purified by molecular sieve and partition chromatography. The analogs themselves possess biological activity (as assayed for effects on mean arterial blood pressure and isolated rat uterus). [4-Br-Phe8]-BK was 1.5 to 3 times as active as bradykinin. [4-Br-Phe5,8]-BK was approx. 22% as active as BK and [4-Br-Phe5]-BK was approx. 18% as active. [4-Br-Phe5]-BK was submitted to catalytic dehalogenation with 10% Pd/C and 5% Rh/CaCO3 in H2O and DMF (1:1) plus 10 Ci of 3H2. [4-3H-Phe5]-BK was obtained at 6.7 Ci/mmole in an overall yield of 15%. [4-3H-Phe8]-BK was prepared similarly to yield an intrinsically-labelled peptide with a specific radioactivity of 21 Ci/mmole.

PULMONARY MICROVASCULAR OCCLUSION IN AGGREGATE ANAPHYLAXIS

The acute respiratory distress syndrome (ARDS) is a common late stage manifestation of several different kinds of severe lung injury (1). A priori, there is little reason to believe that the early pathogenesis of, e.g., sepsis-related lung injury is identical, or even similar, to the early pathogenesis of lung injury caused by aspiration of gastric contents, pneumonia or microthromboembolism even though all causes of serious diffuse lung injury may lead to a common clinical presentation. Thus, it is timely to examine separately the multiple causes of lung injury with the goal of developing appropriate means of treatment of each so as to prevent progression of the lung injuries to frank ARDS.