Faculty Bibliography

We have investigated methods of stabilizing prolidase by chemical modification and covalent coupling to various supports, for use in protein hydrolysis and possible use in enzyme replacement therapy. Purified acetone powder of calf brain prolidase was further purified by gel filtration on Sephadex G-200 and chromatography on DEAE-Sephadex A25. Polyacrylamide gel electrophoresis showed that the number of bands was reduced from 11 to 2. Since yields were low, the purified (NH4)2SO4 fraction was used in all experiments. Thiolation of the enzyme reduced the amount of protein coupled to AH- or CH-Sepharose 4B. Activities were highest when the protein was linked through its carboxyl groups. The coupled enzyme showed much greater thermal stability than its free counterpart. Of the bound preparations, the thiolated was less stable than the untreated. Untreated and thiolated enzymes bound to either matrix showed higher activity at low pH and less at high pH than the free material. Thiolation shifted the pH maximum from 6.8 to 7.5. The free thiolated enzyme and that bound to activated SH-Sepharose 4B showed greater thermal stability and a broader pH range of optimal activity than the bound untreated enzyme. These results show that prolidase can be immobilized by coupling to an insoluble matrix through various types of covalent bonds with retention of activity and increased stability

The reaction of purified rat liver phenylalanine hydroxylase (PheH) with a variety of diazonium, monofunctional and bifunctional reagents or covalent coupling to several polyamino acid and dextran supports resulted in no improvement in stability

The best from a series of matrices for the covalent coupling of rat liver phenylalanine (Phe H) was activated thiol-Sepharose 4B. An average of 0.38 mumoles of enzyme thiol was coupled per mumole of matrix thiol, representing 21 mg of protein per g of carrier. After thiolation with N-acetylhomocysteine thiolactone the enzyme, bound to the same matrix, was thermally more stable with a broad pH range of optimal activity. Thiolation with S-acetylmercaptosuccinic anhydride markedly reduced hydroxylase activity whereas succinylation with succinic anhydride had little or no effect. Treatment of the thiolated enzyme with various oxidants to form disulfide bridges did not increase the thermal stability. Efforts to improve stability by formation of new linkages while the enzyme was still bound to the matrix were unsuccessful. The untreated or thiolated enzyme was inactivated upon reaction with glutaraldehyde

We investigated whether the higher rate of amino acid incorporation into immature than into mature brain protein is due to (a) rapid growth, (b) a small rapidly metabolized protein pool, or (c) a higher turnover rate of most of the protein. We measured net growth and the incorporation of [14C]tyrosine or [14C]valine into brain proteins in young rats and mice. The specific activity of the free amino acid pool was kept constant in the tyrosine experiments. Incorporation of tyrosine into protein was continued for up to 30 h by which time the specific activity of protein-bound amino acid reached 1/3 of that of the free (precursor) amino acid. The growth (accretion) of brain proteins was approx. 0.635% per h in mice and rats in the 1-4 day period after birth. In previous studies we found that the turnover rate of the bulk (about 96%) of adult brain proteins is below 0.3% per h. Because of the presence of a small (about 4%) active pool the average turnover rate is 0.6% per h. The present experiments show a degradation rate of 0.7-1.1% per h in the brain proteins of the young. This high metabolic rate is not due to a small rapidly degraded fraction of protein. The very rapid protein fraction previously seen in adult rats is either very small (below 1%) or absent in the young. Thus most of the proteins in the immature brain during the rapid growth phase are formed and broken down at a rate that is approximately three times higher than that of the bulk of proteins in the adult brain. The small active protein pool in the adult on the other hand has a metabolic rate higher than that of the immature brain proteins