Frank F. Davis

Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to bovine liver arginase using 2,4,6-trichloro-s-triazine as the coupling agent. The conjugate (PEG-arginase), with PEG attached to 53% of the amino groups, retained 65% of its original enzymatic activity. Mice were injected intravenously with arginase or PEG-arginase for periods of one to three months. The blood-circulating life of PEG-arginase was greatly extended over that of arginase. The half-life of injected arginase at day 30 was less than 1 h, whereas that of the PEG-enzyme was 12 h. Antisera from mice injected with native arginase reacted against arginase but not against PEG-arginase when tested by immunodiffusion. Antisera from animals injected with PEG-arginase reacted neither with native arginase nor PEG-arginase. The data indicate that arginase modified by PEG has been rendered both non-immunogenic and non-antigenic when tested in mice. The injection of PEG-arginase into mice did not induce tolerance toward the native enzyme. Injected PEG-arginase, in the presence of precipitating antibody directed against native arginase, circulated at the same level as in virgin animals. The attachment of PEG to arginase altered its kinetic properties.

Properties of two urate oxidases modified by the covalent attachment of poly(ethylene glycol)

Poly(ethylene glycol) of 5 000 daltons has been attached covalently to preparations of urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from hog liver and Candida utilis. Attachment of sufficient poly(ethylene glycol) to either urate oxidase renders the enzyme incapable of eliciting antibody production in mice, or of reacting with antibodies to the unmodified enzyme. The poly(ethylene glycol) : urate oxidase conjugates exhibit higher Km and lower V values than the unmodified urate oxidases. Optimal pH values are increased for the poly(ethylene glycol) : urate oxidases, and optimal temperatures are decreased. The blood circulating lives of the modified urate oxidases following intravenous injection are much longer than those of the unmodified urate oxidases: repetitive injections over a period of 90 days dd not alter the blood circulating lives of the poly(ethylene glycol) : urate oxidases. The unmodified enzymes, on the other hand, were cleared from the blood with extreme rapidity after a few intravenous injections.

Preparation and properties of polyethylene glycol-trypsin adducts.

The covalent attachment of polyethylene glycol of 5000 daltons to non-essential groups on trypsin produces an adduct that no longer precipitates with anti-trypsin antibody. In comparison with trypsin, polyethylene glycol-trypsin preparations show equal or greater activity against N-alpha-benzoyl-L-arginine ethyl ester, about one-fourth activity against angiotensin II, and little activity against bovine liver catalase. The polyethylene glycol-trypsin adduct dissolves soft blood clots at one-fourth the rate of trypsin. Soybean trypsin inhibitor produces two-thirds inhibition of the adduct under conditions that cause complete inhibition of trypsin.

Induction of tolerance in mice by uricase and monomethoxypolyethylene glycol-modified uricase

The ability to induce tolerance to uricase by the administration of native uricase, and uricase modified by the covalent attachment of monomethoxypolyethylene glycol (PEG) was examined. Uricase, and uricase with PEG attached to 35% (PEG-uricase 35%) and 70% (PEG-uricase 70%) of available amino groups were found to induce tolerance in mice not previously sensitized to uricase. There was a dampening of the IgG, IgE and IgM antibody response to uricase which persisted even after a second sensitizing dose of uricase was administered to these animals. PEG-uricases were found to have little or no immunogenicity when injected into mice and a reduced immunogenicity and antigenicity when tested in rabbits. Native uricase, however, was found to be immunogenic and antigenic in mice and rabbits. Mice sensitized to native uricase were injected with uricase, PEG-uricase 35% or 70% to induce tolerance. After a second sensitizing injection of uricase, circulating levels of IgE, IgG and IgM were measured. All three enzymes induced tolerance in the IgE class of antibody but there was no significant change in the hemagglutinating antibody levels of the mice. Mice injected with 1 mg of uricase died from anaphylaxis. PEG-uricase 35% was found to induce the most effective tolerance in both unsensitized and sensitized mice.

Enzyme-Polyethylene Glycol Adducts: Modified Enzymes with Unique Properties

There are many clinical disorders which, in theory might be treated by the use of appropriate enzymes. However, there are serious problems associated with enzyme therapy. First, inadequate amounts of human enzymes are available; and most foreign enzymes are immunogenic in humans. They can be administered only a few times. Second, the circulating life in the blood may be very short even on the first injection (often a matter of a few minutes). And third, the cost of highly purified enzymes may be too great to allow general use.

Polyoxyethylated cholesterol derivatives Organic synthesis, cellular uptake and effect on lipid metabolism in cultured skin fibroblasts

Abstract Polyoxyethylated derivatives of cholesterol were synthesized by adduction of ethylene oxide to the 3-β-hydroxy position of cholesterol. Derivatives with the methoxy terminal were prepared by condensing methoxypoly(oxyethylene)methane sulfonate with cholesterol. Derivatives with nine ethoxy groups were isolated by the combined use of high pressure liquid chromatography and thin-layer chromatography and identified by proton magnetic resonance. In cultured human skin fibroblasts, uptake of polyoxyethylated [ 3 H]cholesterol was linear up to a concentration of 60 μM. Uptake at 10 μM concentration was linear for 5 h and amounted to 2.5% of the total added label per h. All of the cellular radioactivity was recovered in polyoxyethylated cholesterol and no label appeared in other lipid fractions. Polyoxyethylated cholesterol (10 μM) inhibited the incorporation of acetate into cholesterol and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, 50% inhibition occurring at 1 and 3 h, respectively. After 18 h incubation, 50% inhibition of both activities occurred at 5 μM and near total inhibition at 25 μM. The incorporation of leucine into protein, uridine into RNA and thymidine into DNA were not affected significantly. Methoxypolyoxyethylated cholesterol gave similar results. There were no differences between normal and homozygous familial hypercholesterolemic fibroblasts which lack membrane receptors for low density lipoproteins. Incorporation of acetate into fatty acids was suppressed by polyoxyethylated cholesterol but not by 25-hydroxycholesterol. Polyoxyethylated cholesterol had no effect on the activity of fatty acid synthetase. Efflux of polyoxyethylated cholesterol from cells had a rapid phase amounting to 55% in the first 2 h, followed by a slow leakage of 15% in the next 20 h. Recovery of the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was faster than that of incorporation of acetate to cholesterol and fatty acids but all these activities remained below the control levels, suggesting that the polyoxyethylated cholesterol retained within the cell continues to exert its inhibitory effect on lipid synthesis.

Studies on pseudouridylic acid synthetase from various sources

Abstract Pseudouridylic acid synthetase (ΨMP-synthetase) was investigated in various tissues. 1. 1. Normal tobacco stem tissue contains ΨMP-synthetase which elutes from a DEAE-cellulose column in 0–0.1 M NaCl. 2. 2. Tobacco stem tissue transformed with Agrobacterium tumefaciens contains a ΨMP-synthetase that also elutes from the DEAE-cellulose column in 0–0.1 M NaCl. 3. 3. A. tumefaciens ΨMP-synthetase elutes under similar chromatographic conditions at about 0.4 M NaCl. 4. 4. It is concluded that A. tumefaciens ΨMP-synthetase is not synthesized in the transformed plant cell. 5. 5. ΨMP-synthetase, uridine ribohydrolase (EC 3.2.2.3), and ribonuclease activities were determined in a number of other sources.

A Rapid Purification Procedure for L-Asparaginase from Vibrio Succinogenes

A simple procedure has been developed for the purification of L-asparaginase from Vibrio succinogenes. Only two steps of ion-exchange chromatography are required. A higher yield and higher specific activity are obtained than previously reported.

Effect of cycloheximide on the synthesis and modification of ribosomal RNA in Neurospora crassa.

Abstract 1. 1. Extraction with hot phenol revealed that in Neurospora crassa grown in the presence of cycloheximide a high molecular weight ribosomal precursor RNA accumulates. 2. 2. This RNA is partially methylatable, and both methyl and uracil labels can be chased into rRNA on reincubation of cycloheximide-treated cells in antibiotic-free medium. 3. 3. Methylation of RNA was uniformly and substantially decreased in the methylated nucleotides which were examined. By contrast, the pseudouridylation process in rRNA and ribosomal precursor RNA was unaffected by cycloheximide.

Barrier electrophoresis: A new electrophoretic technique☆☆☆

Abstract Barrier electrophoresis, a preparative technique that combines the resolving capacity of zone electrophoresis in gels with continuous flow, is described. Several applications of barrier electrophoresis to the purification of macromolecules are presented.