Irwin B. Wilson

Enzymic parameters: measurement of V and Km.

Abstract A slight modification of the Lineweaver—Burk equation (and other variants) in which the form is retained but the instantaneous velocity is replaced by the negative change in substrate concentration divided by the time and the initial substrate concentration is replaced by the arithmetic average substrate concentration during the time interval, allows this linear reciprocal form to be used with little error even when as much as 50% of the substrate is consumed. This result is in sharp contrast to the prevailing belief that the initial velocities and initial substrate concentrations must be used.

A simple spectrophotometric assay for amino acyl arylamidases (naphthylamidases, aminopeptidases)

Abstract A simple spectrophotometric assay of amino acyl arylamidases (naphthylamidases, aminopeptidases) has been developed based upon the difference spectrum of the substrate and the enzymic hydrolysis product, β-naphthylamine. A maximum difference in the molar extinction coefficients occurs at 340 mμ; Δϵ = 1780, pH > 5.0. The assay is very simple, and is rapid, sensitive, and suitable for recording. The method provides a means for the determination of enzymic activity as well as for a study of kinetic constants. The method is applicable to the study of different arylamidases using different amino acid amides of β-naphthylamine.

Human plasma converting enzyme

Abstract Angiotensin-converting enzyme was partially purified from human plasma by ammonium sulfate and Sephadex gel nitration. Some of its kinetic and physiological properties were discussed in comparison with those of hog plasma-converting enzyme. The K m value for human plasma converting enzyme was obtained as 4.5 × 10 −5 m . The S 20,W value (8.2) for the enzyme was obtained by the sucrose density gradient sedimentation technique and the same molecular weight for the converting enzyme in human and hog plasma was estimated to be about 150,000 daltons. The maximum activity of the enzyme in human plasma is about 4.5 units per ml, and the calculated half-time for the conversion of angiotensin I to angiotensin II is about 7 min. This slow rate, as in hog plasma, suggests that there must be more to the “activation” of angiotensin than is immediately obvious.

Angiotensin-converting enzyme from guinea pig and hog lung.

Abstract Lung contains a true angiotensin-converting enzyme, i.e., an enzyme that converts angiotensin I to angiotensin II by the hydrolytic removal of the dipeptide histidylleucine. The soluble enzymes from guinea pig and hog lung were purified more than 200-fold. These enzymes are quite similar in having the same Km, 20 μM, the same s20,w value of 8.2 from sucrose gradient measurements, and the same Stokes radius from Sephadex G-150 gel filtration, corresponding to a molecular weight of about 150 000 d. The plasma enzymes have the same s20,w values and Stokes radii as the lung enzymes; the Km values are similar but slightly higher 40 μM. In contrast to plasma, the lung, especially hog lung, contains a considerable amount of histidylleucine hydrolyzing activity. This enzyme was obtained in a partially purified form with a specific activity of 1.6 μmoles/min per mg protein. The molecular weight of histidylleucine cleaving enzyme was estimated to be about 80 000 based on the corrected s20,w value of 5.3 S and the relative elution volume from gel filtration. The Km value of 200 μM for histidylleucine was calculated. The maximum tissue converting enzyme activities of guinea pig and hog that we obtain are about 30 and 10 nmoles/min per g wet tissue. This amount is less than we expected since in the dog more than 50% of angiotensin I is converted to angiotensin II in a single passage through the lung.

Purification of human serum angiotensin I-converting enzyme by affinity chromatography

Abstract A relatively simple procedure is described for purifying human serum angiotensin-converting enzyme. The enzyme was purified 130,000-fold to electrophoretic homogeneity using affinity chromatography as the principal purification step. The ligand was an immobilized competitive inhibitor, d -cysteinyl- l -proline. A six-carbon spacer arm was satisfactory for trapping the enzyme; 80% of the bound enzyme was eluted with 3 m urea-1.0 m NaCl-0.1 m Tris, pH 8.3. The specific activity was 39 units/mg protein and the molecular weight (155,000), isoelectric point (4.7), kinetic properties, and the effect of various inhibitors are in agreement with published reports.

Inhibition and affinity chromatography of human serum angiotensin converting enzyme with cysteinyl-proline derivatives.

Abstract Several structural derivatives of the dipeptide, l (and d )-cysteinyl- l -proline were synthesized and shown to be very potent competitive and noncompetitive inhibitors of human serum angiotensin I-converting enzyme. Only if the sulfhydryl group of the cysteine was blocked with benzyl, trityl, or benzyloxycarbonyl protecting groups, was the dipeptide a noncompetitive inhibitor. Compounds with free sulfhydryl groups were competitive inhibitors with K i values in the 10 −8 m range. d -Cys- l -Pro, our most potent inhibitor ( k 1 = 0.0055 μM), was an order of magnitude more potent than l -Cys- l -Pro consistent with findings of Cushman et al. (1977, Biochemistry 16 , 5484) that -CH 3 group substitution improves binding if the configuration is d but diminishes binding if the configuration is l . Zinc and calcium ions released inhibition by some of the noncompetitive, but only one, of the competitive inhibitors. The noncompetitive inhibitor, l -cysteinyl(benzyl)- l -proline, and the competitive inhibitor, l -cysteinyl- l -proline, were used as affinity ligands to obtain near homogenous (25 units/mg) enzyme from human plasma. The observation that compounds with a free sulfhydryl group are competitive inhibitors and those in which the sulfhydryl groups are blocked are noncompetitive inhibitors can be rationalized if the active site of the converting enzyme is an extended linear trench.

The inhibition of cholinesterase by diethyl phosphorochloridate

Abstract A method is described which enables the measurement of the rate constant for the inhibition of enzymes by covalent inhibitors that hydrolyze rapidly. The method(s) also yields the rate constant for the hydrolysis of the inhibitor. It was found that diethyl phosphorochloridate, depending on the buffer composition, hydrolyzes with a first-order rate constant of 1.0 min−1 in phosphate buffer ( I = 0.1 ) at pH 7.0 and 25 °C and inhibits acetylcholinesterase (eel) (acetylcholine hydrolase, EC 3.1.1.7) with a second-order rate constant of 1.5·106 M−1·min−1. Cholinesterase from bovine erythrocytes and horse serum were also studied. In any case, the phosphorochloridate was not found to be much more potent as an inhibitor than the corresponding phosphorofluoridate, even though the rate of hydrolysis of the latter is 2000 times slower.

Angiotensin-converting enzyme from porcine plasma

Abstract Angiotensin-converting enzyme was purified from porcine plasma by (NH 4 ) 2 SO 4 fractionation, Sephadex G-200 gel filtration and DEAE-cellulose chromatography. These techniques combined to give a preparation which hydrolyzed 50 nmoles of substrate per mg per minute. This preparation is considerably more purified than previously reported preparations. The radioactive assay of Huggins was modified in that high voltage electrophoresis was used to separate histidylleucine from the substrate. The K m value for the enzyme was obtained as 4.2·10 −5 M. The enzyme reaction was inhibited by angiotensin II, but not by histidylleucine. The s 20 , w value (8.3) of the converting enzyme was obtained by the sucrose density gradient sedimentation technique and the molecular weight was estimated to be 155 000. The maximum activity of hog plasma is about 9 nmole per ml per minute. The calculated half time for the conversion of angiotensin I to angiotensin II in plasma is about 3 min. This slow rate suggests that there must be more to the “activation” of angiotensin than is immediately obvious and in this sense is consistent with the conclusion of Ng and Vane 4–6 that the lung, and not the blood, is the primary site of conversion of angiotensin I to angiotensin II.

Atrial tissue contains a metallo dipeptidyl carboxyhydrolase not present in ventricular tissue: Partial purification and characterization

A new membrane-bound dipeptidyl carboxyhydrolase has been identified in bovine atrial tissue, and has been partially purified after extraction with Triton X-100. This enzyme, found in quantities of 0.01-0.03 units/g tissue assayed with Bz-Gly-His-Leu, is potentially capable of hydrolyzing atriopeptin II to atriopeptin I. The enzyme is located in the microsomal fraction and in sucrose density fractions enriched for atrial granules. The enzyme is completely inhibited by reagents for heavy metals such as EDTA, o-phenanthroline, dithiothreitol, and mercaptoethanol. The latter two compounds are also disulfide reagents. The atrial enzyme is also inhibited by D-2-methyl-3-mercaptopropanoyl-L-Pro(Captopril), 3-mercaptopropanoyl-L-Pro, 2-D-methylsuccinyl-L-Pro, and bradykinin potentiating factor, all inhibitors of the angiotensin I-converting enzyme. However, the atrial enzyme differs from the converting enzyme in a number of kinetic and molecular properties. Its activity increases with ionic strength, but the atrial enzyme does not have a chloride dependence for Bz-Gly-His-Leu hydrolysis; the pH optimum, 7.3, is slightly lower, and it is 5500 times less sensitive to the very potent converting enzyme inhibitor, D-Cys-L-Pro. The strokes radius of the atrial enzyme is 5.00 nm as compared to 4.10 nm, and its molecular weight is 240,000 compared to 145,000. Ventricular tissue, which does not contain the atrial peptides, does not contain the dipeptidyl carboxyhydrolase enzyme.

Synthesis of tert-butyl aminocarbonate, a new type of compound that can be used to acylate amines

Abstract tert-Butyl aminocarbonate (tert-butyloxycarbonyloxyamine) was prepared by reaction of hydroxylamine with excess di-tert-butyl dicarbonate. Amino carbonates have not previously been described. This compound rapidly acylates amines in both organic and aqueous solutions.