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P M Horowitz

Publications and source records attributed to P M Horowitz.

150 records · Page 9Linked to original sources
PubMed1976

A reexamination of the postulated charge transfer interactions at the active site of the enzyme rhodanese.

Spectral and kinetic studies of the interaction of N-methylnicotinamide chloride and nicotinamide with the enzyme thiosulphate sulphurtransferase (thiosulphate: cyanide sulfurtransferase, EC 2.8.1.1) (also known as rhodanese) have been performed and compared with previous inhibition data obtained with N-1-(4-pyridyl)pyridinium chloride (NPP). Like NPP both N-methylnicotinamide chloride and nicotinamide are competitive inhibitors of rhodanese with respect to the substrate thiosulfate. Rhodanese binding of N-methylnicotinamide chloride gives rise to no charge transfer absorbtion band. In addition, the free energy of interaction (deltaG0) of NPP with rhodanese is approximately equal to the sum of the individual deltaG0 values of MNA and NA. These compounds are analogous to the two halves of the NPP structure. We conclude that NPP and N-methylnicotinamide chloride are not bound via a charge transfer mechanism. The major stabilizing influence appears to be an ionic interaction with an anionic enzyme site with accessory apolar stabilization. It is postulated that the ionized active site sulfhydryl group in rhodanese could provide the ionic site.

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PubMed1976

A study of the single polypeptide nature of rhodanese. A comparison of different preparations.

The enzyme rhodanese (EC 2.8.1.1) appears as a single polypeptide chain protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this species is approx. 33 000. This contrasts with previous reports that rhodanese behaves on gel filtration chromatography as a rapidly equilibrating monomer-dimer system composed of identical subunits with a molecular weight of 18 500. We have investigated this apparent discrepancy by isolating the enzyme by the two different preparative procedures used in the above investigations. The two crystalline samples were subjected to gel filtration chromatography under a wide variety of conditions and to sodium dodecyl sulfate disc gel electrophoresis. The two preparations yielded rhodanese which behaved identically and no evidence for the monomeric species was obtained under any experimental condition tested. Thin-layer gel chromatography of clarified liver homogenates gave no evidence of rhodanese species other than that present in the purified samples. The variation in molecular weights observed in gel filtration chromatography may be a reflection of the conformational mobility of the enzyme leading to solvent-dependent changes in Stokes radius. If rhodanese is dimeric, special interactions must stabilize it under the conditions tested here.

Animals↗
PubMedDate not supplied

Purification of bovine liver rhodanese by low-pH column chromatography.

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.

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