Barton Holmquist

A continuous spectrophotometric assay for angiotensin converting enzyme

Abstract Furanacryloyl tripeptides conforming to the known substrate specificity of the angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1) have been employed to provide a continuous spectrophotometric assay for this peptidase in the visible region. The assay is based on a blue shift of the absorption spectrum that occurs upon hydrolysis of the substrate to produce a furanacryloyl-blocked amino acid and a dipeptide. Of the various furanacryloyl tripeptides tested, furanacryloyl- l -phenylalanylglyeylglycine exhibits the most suitable characteristics for routine assays of angiotensin converting enzyme.

Thermolysin: A zinc metalloenzyme

Metal analyses and inhibitor studies have shown that thermolysin, a neutral protease from B. thermoproteolyticus, is a zinc metalloenzyme. The relevance of this finding to the active site characteristics of other bacterial neutral proteases and to those of alkaline proteases is considered.

Neocarzinostatin: Spectral characterization and separation of a non-protein chromophore

Abstract Chromatographically purified neocarzinostatin exhibits absorption, fluorescence, magnetic circular dichroic and circular dichroic spectral characteristics above and below 300 nm atypical for a protein with its reported aminoacid composition, indicating the presence of a non-protein chromophore. The drug complex, stable at acidic pH, can be dissociated by treatment with reducing or denaturing agents at neutral or basic pH. Chromatography of the dissociated complex, or more conveniently, methanol extraction of the lyophilized drug, separates a protein with an amino-acid composition identical to neocarzinostatin and a highly fluorescent chromophore free of amino-acids.

[2] Elimination of a metals

Publisher Summary This chapter discusses the elimination of adventitious metals. The two methods reported in this chapter, dithizone extraction and ion exchange with a chelating resin, have been used extensively in numerous laboratories and have proven to be necessary in all aspects of work, involving the replacement or depletion of the metals in metalloenzymes. Diphenylthiocarbazonc (dithizone) is a complexing agent soluble in organic solvents that reacts with various metals to form organic soluble chelates. Upon rectal complexation, the bright green color of its solutions turns red, the color change serving as a sign of metal contamination. It is stressed that nothing supplants the verification of any procedure used for the elimination of adventitious metal. Hence, actual metal analysis of solutions, using methods, such as atomic absorption spectroscopy, is desirable subsequent to metal extraction. It is very easy to recontaminate solutions or to have such procedures go awry; therefore, if one or more metals are of particular concern, its presence or absence should be verified when possible.

Functional residues at the active site of angiotensin converting enzyme

Abstract A series of chemical modification reactions have been carried out with rabbit pulmonary angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1) in order to identify amino acid residues essential for its catalytic activity. The enzyme is rapidly inactivated by nitration with tetranitromethane and by O-acetylation with N-acetylimidazole. Deacylation with hydroxylamine restores activity to the acetylated enzyme, while the inhibitor, β-phenylpropionyl-L-phenylalanine, protects against acetylimidazole inactivation. These results indicate the presence of functional tyrosyl residues at the active site of the enzyme. Reaction with butanedione decreases activity, an effect that is markedly enhanced by the presence of borate, indicating essential arginyl residues. In addition, activity is diminished by the carboxyl reagent, cyclohexylmorpholinoethyl carbodiimide. Thus, the three functional residues long known to be components of the active site of bovine carboxypeptidase A, tyrosyl, arginyl, and glutamyl, have counterparts in the angiotensin converting enzyme. The effects of pyridoxal phosphate and a number of other reagents demonstrate that the converting enzyme also contains an important lysyl residue.

Human liver class III alcohol and glutathione dependent formaldehyde dehydrogenase are the same enzyme

Human liver class III alcohol dehydrogenase (chi chi-ADH) and glutathione dependent formaldehyde dehydrogenase are the same enzyme. The enzyme, chi chi-ADH, exhibits a kcat of 200 min-1 and a km of 4 microM for the oxidation of formaldehyde, but only in the presence of GSH. In the absence of GSH the enzyme is essentially inactive toward formaldehyde but very active toward long chain alcohols. Thus, as in the rat (Koivusalo, M., Baumann, M., and Uotila, L. (1989) FEBS Letters 257, 105-109), the class III alcohol dehydrogenase and the GSH dependent formaldehyde dehydrogenase are identical enzymes. S-Hydroxymethyl derivatives of 8-thiooctanoate and lipoate are also very active substrates. The activity is specific for class III alcohol dehydrogenase; neither the class I and II nor the horse EE, ES, and SS isozymes oxidize hemithiolacetals. o-Phenanthroline competitively inhibits both activities and the two substrate types compete with each other.

Fluorometric assays for isozymes of human alcohol dehydrogenase

Two new fluorogenic substrates for human alcohol dehydrogenase (ADH), 4-methoxy-1-naphthaldehyde (IA) and 6-methoxy-2-napthaldehyde (IIA), are described. The 2-naphthaldehyde derivative fluoresces in aqueous media with a quantum yield of 0.22 with an emission maximum at 450 nm, but the 1-naphthaldehyde shows only weak fluorescence. The corresponding alcohol reduction products, 4-methoxy-1-naphthalenemethanol (IB) and 6-methoxy-2-naphthalenemethanol (IIB), exhibit fluorescence in the near uv region with quantum yields of 0.36 and 0.26, respectively. The Km values for the individual homogenous class I ADH isozymes, with the above naphthaldehydes as substrates, range from 0.35 to 11.5 microM. The kappa cat values range from 70 to 610 min-1 and are thus comparable to those for the best ADH substrates. Except for the beta 1 beta 1 isozyme, IA is the preferred substrate for class I ADH isozymes while IIA is rapidly reduced by class II (pi-ADH). The sensitivity and specificity of the enzymatic assay with IA as substrate are demonstrated and provide the basis for the determination of class I ADH activity in human serum.

cDNA sequence of human class III alcohol dehydrogenase

A human placental cDNA library was screened using oligonucleotide probes based on the peptide sequence of the human class III alcohol dehydrogenase. An incomplete cDNA clone covering most of the coding sequence of class III alcohol dehydrogenase was isolated from a human placental cDNA library. This was subsequently used as a probe to obtain a full-length clone from a human testicular library. The cDNA sequence codes for a protein that is identical to the enzyme purified from human liver. Southern analysis of human genomic DNA suggests that it may contain more than a single copy per haploid genome.

One hundred fold increased activity of Aeromonas aminopeptidase by sequential substitutions with Ni(II) or Cu(II) followed by zinc

Full substitution of Cu(II) or Ni(II) for the two g-atom zinc in Aeromonas aminopeptidase hyperactivates the enzyme 6.5 and 25 fold respectively. Even greater enhancements of activity can be achieved with mixed metal substitutions. Thus, apoenzyme reactivated by first adding one g-atom zinc followed by one g-atom of either Cu(II) or Ni(II) is 15 and 22 times more active than the native enzyme. Reversing the order, i.e. by first adding either one g-atom Cu(II) or Ni(II) followed by one g-atom zinc, activates the enzyme nearly 100 fold. The order of metal addition is critical and suggests the existence of two non-identical metal sites, each with a different function.

Structural and electronic mimics of the active site of cobalt(II)-substituted zinc metalloenzymes

Complexes of cobalt(II) and zinc(II) which involve monodentate coordination of two alkyl carboxylate and two imidazole ligands in a slightly distorted tetrahedral fashion have visible and magnetic circular dichroism spectra remarkably similar to the cobalt(II)-substituted proteolytic enzymes thermolysin and carboxypeptidase A. Single crystal x-ray structure determinations on [Co(C2H5COO)2Im2], Im = imidazole, and its zinc counterpart reveal only minor structural differences between the cobalt and zinc species. Electron paramagnetic resonance spectra of cobalt(II) doped into zinc(II) complexes with known structures demonstrate the extreme sensitivity of the g-values to minor structural differences.