Ernst A. Noltmann

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase.

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO2 hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a Km of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (Ki = 1.22 × 10−3M), arsenate (Ki = 1.17 × 10−3M), and molybdate (Ki = 1.34 × 10−7M), with these inhibitors having no effect on the CO2 hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO2 hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO2 hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.

Basic muscle protein, a third genetic locus isoenzyme of carbonic anhydrase?

Abstract Rabbit muscle cytosol extract contains a basic protein which represents about 2% of the total cytosol protein. It contains zinc in a 1:1 stoichiometric ratio, based on a molecular weight of 30,000, and it catalyzes the hydration of CO 2 . It is immunochemically distinct from the high and low activity forms of rabbit blood carbonic anhydrase. It has comparatively poor activity as an esterase, and about 20% of the CO 2 hydratase activity of the rabbit blood low activity carbonic anhydrase. This CO 2 hydratase activity is not inhibited by acetazolamide at concentrations which totally inhibit the activity of the blood carbonic anhydrases. The evidence obtained to date, though circumstantial, suggests that this basic metalloprotein is a carbonic anhydrase derived from a third genetic locus with properties considerably different from those of the mammalian carbonic anhydrases heretofore identified.

A rapid pH-stat assay for phosphofructokinase and the forward reaction of phosphoglucose isomerase

Abstract Continuously recording pH-stat assays are deseribed for phosphofructokinase and the forward reaction of phosphoglucose isomerase. The validity of the assays is discussed in terms of stoichiometry, linearity, accuracy, and possible interference from undesired side reactions.

Arginine-specific modification of rabbit muscle phosphoglucose isomerase: Differences in the inactivation by phenylglyoxal and butanedione and in the protection by substrate analogs

Rabbit muscle phosphoglucose isomerase was modified with phenylglyoxal or 2,3-butanedione, the reaction with either reagent resulting in loss of enzymatic activity in a biphasic mode. At slightly alkaline pH butanedione was found to be approximately six times as effective as phenylglyoxal. The inactivation process could not be significantly reversed by removal of the modifier. Competitive inhibitors of the enzyme protected partially against loss of enzyme activity by either modification. The only kind of amino acid residue affected was arginine. However, more than one arginine residue per enzyme subunit was found to be susceptible to modification by the dicarbonyl reagents. From protection experiments it was concluded (i) that both modifiers react specifically with an arginine in the phosphoglucose isomerase active site and nonspecifically with one or more arginine residues elsewhere in the enzyme molecule, (ii) that modification at either loci causes loss of catalytic activity, and (iii) that butanedione has a higher preference for active site arginine than for arginine residues outside of the catalytic center whereas the opposite is true for phenylglyoxal.

Purification and properties of pig muscle carbonic anhydrase III

Pig muscle carbonic anhydrase III (carbonate hydro-lyase, EC 4.2.1.1) has been isolated and purified to homogeneity with chromatographic techniques. It has been found to be a 30 kDa protein displaying the same three activities (CO2 hydratase, acetate esterase, p-nitrophenyl phosphatase) previously described for the rabbit muscle isoenzyme, including the phosphatase activity not seen in the erythrocyte isoenzymes. The turnover numbers of the three activities are of the same order of magnitude as previously reported for rabbit muscle carbonic anhydrase III. Km and Vmax for the pig muscle CO2 hydratase activity were found to be 83 mM and 6000 s-1, respectively. The extinction coefficient at 280 nm (1 cm light path) is 22.2 for a 1% solution. Five half-cystine residues determined by performic acid oxidation are free for reaction with p-mercuribenzoate but only four are accessible to titration with dithiobisnitrobenzene. The amino acid composition of the pig muscle isoenzyme III has a high level of homology compared with that of rabbit and bovine muscle carbonic anhydrases III.

Molecular characterization of pig muscle phosphoglucose isomerase

Abstract As a corollary to X-ray crystallographic work performed by H. Muirhead, detailed studies on crystalline pig muscle phosphoglucose isomerase have been conducted to establish its basic physical and chemical properties. The enzyme species being investigated by X-ray diffraction has been determined to be isoenzyme III. Its molecular weight in the native state was found to be 132,000, its s020,w value to be 7·25 S. The enzyme is composed of two subunits of equal molecular weight (66,000). Its amino acid composition is largely similar to that of rabbit muscle phosphoglucose isomerase, with the significant exception that the pig muscle isomerase contains only three sulfhydryl groups per polypeptide chain (two of them accessible to titration with p-mercuribenzoate) as compared with twice that number for the rabbit muscle enzyme. This low number of sulfhydryl groups is interpreted as being responsible for the ease with which heavy-atom, isomorphous derivatives could be prepared for the pig muscle enzyme by Shaw & Muirhead (1977).

Identification of pseudoisoenzymic subforms of muscle carbonic anhydrase

Rabbit muscle carbonic anhydrase III, a recently discovered third isoenzyme (possibly muscle specific) of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) (Register, A.M., Koester, M.K. and Noltmann, E.A. (1978) J. Biol. Chem. 253, 4143--4152) has been subjected to isoelectric focusing. When monomer samples, shown to be homogeneous by both ion-exchange and molecular sieve chromatography, were analyzed by this technique, three subspecies were produced, which were similar in amino acid composition and specific CO2 hydratase activity. In addition to having either monomer or dimer status, the subspecies differed in the extent of oxidation of their sulhydryl groups and in their isoelectric pH values (9.3, 8.8, and 8.4, respectively). Also, the presence of dithiothreitol will affect their relative concentrations. These subforms are therefore designated as pseudoisoenzymes and are considered to be neither genetically nor functionally separate enzyme species.

Chromatographic identification of a new subform of carbonic anhydrase in rabbit erythrocytes

Abstract In addition to the two forms of carbonic anhydrase (CA) known to occur in rabbit erythrocytes, a third enzyme species has been isolated and partially characterized. The three Chromatographic forms elute from diethylaminoethyl-Sephadex roughly in the proportions of 3:5:2, in that order. They do not appear to be interconvertible. Their molecular weights are approximately 30,000. Initial kinetic studies of ester hydrolysis and of CO2 hydration by these carbonic anhydrases identify the first peak to elute as a low-activity form (CA I) and the following two as high-activity forms (CA II, CA II′). Inhibition studies indicate that equal concentrations of all three enzyme species are equally inhibited by equal concentrations of benzenesulfonamide or acetazolamide. The amino acid compositions of the three species support their identification as high- and low-activity forms and indicate substantial differences between CA I and CA II or CA II′, while the latter two differ only slightly. Peptide maps obtained from tryptic digests show considerable differences between the peptides of the high- and low-activity forms, while the peptides of CA II and CA II′ are almost indistinguishable. The available evidence suggests that the newly isolated CA II′ species is a subform of the major high-activity isoenzyme and that a very slight difference in amino acid composition is responsible for a net charge difference between CA II and CA II′, which is reflected in their different Chromatographic properties.

Discovery, characterization, and study of some inhibition properties of two mammalian muscle carbonic anhydrases III.

In a discussion of the muscle isoenzyme carbonic anhydrase 111, it appears to be fitting to begin with a quotation from F. J. W. Roughton’s 1935 paper in Physiological Reviews‘ in which, in the context of refemng to the possible presence of carbonic anhydrase in muscle tissue, he made the statement that it, that is carbonic anhydrase, “would be an enemy of the organism.” In the many studies on carbonic anhydrases over the past 50 years, this statement contributed a great deal to the fact that carbonic anhydrase was claimed to be absent from muscle tissue. The other more tangible feature that prevented for so long the identification of carbonic anhydrase as being present in this tissue was that the classical definition for carbonic anhydrase in a tissue demanded that the production of protons from the reaction between COz and water had to be inhibitable by very low concentrations (lo-’ to lo-* M) of the sulfonamide inhibitor acetazolamide. In addition, investigators working with crude muscle extracts were justifiably concerned about contamination by erythrocyte carbonic anhydrase. Thus, when in the early 1970s our laboratory discovered a homogeneous protein as byproduct of the phosphoglucose isomerase purification procedure from rabbit skeletal muscle (FIG. I),* it took awhile until we convinced ourselves that this protein was indeed a new carbonic anhydrase isoenzyme about which we then reported at the 1977 Federation meetings3 Its primary distinction from the known carbonic anhydrases was indeed that it was inhibited only by very much larger concentrations of acetazolamide M). It was clear that anyone testing a tissue for carbonic anhydrase with the classical criterion that such an enzyme had to be inhibitable by M acetazolamide would conclude that carbonic anhydrase was absent when the inhibition test failed. At about that time, other investigators had also become suspicious of the absence of carbonic anhydrase from muscle tissue. Notably Holmes reported the presence of carbonic anhydrase activity in electrophoretic bands obtained from sheep muscle tissue4 and Tashian’s laboratory observed carbonic anhydrase activity in preparations from bovine skeletal muscle.s to

Large scale isolation of pig muscle phosphoglucose isomerase.

A large-scale purification procedure for phosphoglucose isomerase from pig skeletal muscle is described. It consists of two fractionations by selective precipitation and two ion exchange chromatography steps yielding an end product of approximately 900 units (micromoles of substrate converted to product per min per mg of protein, at 30 degrees) specific activity. The method separates three isoenzymic forms with an overall recovery of about 30% of the original total enzyme activity in the form of Isoenzyme III, the latter being the predominant enzyme species.