Hans C. Rilling

ON THE MECHANISM OF PHOTOINDUCED CAROTENOID SYNTHESIS: ASPECTS OF THE PHOTOINDUCTIVE REACTION.

Abstract Photoinduced carotenoid synthesis has been studied in two species of mycobacteria, Mycobacterium marinum and an unclassified species. The action spectra of photo-induction have been shown to be markedly different for these two organisms. The action spectrum for the Mycobacterium sp. has maxima at 365 and 460 mμ separated by a minimum at 395 mμ. The action spectrum of M. marinum has a principal maximum at 404 mμ and two lesser maxima at 493 and 577 mμ. It is likely that a flavin is the photoreceptor for the Mycobacterium sp. and a porphyrin for M. marinum . The photoinduction of carotenogenesis in Mycobacterium sp. was found to be depressed at acid pH while that of M. marinum was unaffected by that treatment. The photoinductive reaction of both organisms was inhibited by azide at acid pH.

Purification to homogeneity and some properties of squalene synthetase.

Squalene synthetase has been purified to homogeneity from yeast. It is a single polypeptide of Mr 47,000. This enzyme catalyzes the synthesis of squalene from farnesyl diphosphate via presqualene diphosphate. In the presence of reduced pyridine nucleotides, presqualene diphosphate and squalene are produced in a ratio of 6:1 from either the purified protein or the crude microsomal fraction.

Purification and properties of pig liver prenyltransferase: Interconvertible forms of the enzyme☆

Abstract Prenyltransferase (EC 2.5.1.1) has been purified to homogeniety from pig liver. The protein, which has been shown to be homogeneous by electrophoresis under several conditions, has a specific activity fourfold greater than that of previous preparations from this tissue. The protein is a dimer of molecular weight of approximately 80,000 whose subunits were not resolved by electrophoresis in sodium dodecyl sulfate. Michaelis constants for geranyl pyrophosphate and isopentenyl pyrophosphate were determined to be 0.58 and 0.31 μ m , respectively. The homogeneous enzyme could be resolved into two forms by isoelectric focusing or chromatography on DE-52 cellulose under certain conditions. The isoelectric points of the two forms were 4.85 and 4.99. The two forms of the enzyme were interconvertible and probably result from a slow equilibrium between two conformational isomers of the protein.

Determination of isopentenyl diphosphate and farnesyl diphosphate in tissue samples with a comment on secondary regulation of polyisoprenoid biosynthesis

A double-isotope dilution procedure is described for the determination of two isoprenoid precursors, isopentenyl and farnesyl diphosphate. Recovery of each is determined by the addition of the appropriate radioactive diphosphate to the tissue sample. After partial purification, each is coupled by a prenyltransferase with a cosubstrate of known specific activity. The products, doubly labeled farnesyl and geranylgeranyl diphosphates, are cleaved to the parent alcohols by alkaline phosphatase. The resulting polyprenols are isolated by reversed-phase thin-layer chromatography and their radioisotopic content is determined. The levels of these precursors have been measured in livers of rats and mice that have been maintained on several different diets. The concentration of each was about 0.5 mumol/g wet tissue and varied as much as 10-fold under the different test conditions. The levels of isopentenyl diphosphate isomerase, farnesyl diphosphate synthetase, and squalene synthetase were also measured in these animals. The changes in levels of these enzymes, in conjunction with the variation in substrate concentrations, are such that they could substantially influence the rate of cholesterol synthesis in liver.

A STUDY OF INHIBITION OF CAROTENOID SYNTHESIS.

Abstract A Mycobacterium species that synthesizes carotenoids only after light stimulation has been used to study the inhibition of carotenoid synthesis by a variety of compounds. In addition to diphenylamine, which has been shown to inhibit carotenogenesis in many systems, benzophenone, acridine orange, methylene blue, neutral red, toluidine blue, proflavine, acridine, and 9,10-dihydroacridine were found to inhibit the formation of the more highly unsaturated carotenoids. In all instances phytofluene accumulated when the formation of the more highly unsaturated carotenoids was inhibited. Under conditions of high oxygen tension all of these compounds except diphenylamine and benzophenone no longer inhibit carotenogenesis. Molecular models show that the dihydro derivatives of the heterocyclic inhibitors, benzophenone, and diphenylamine have a close structural similarity to carotenoids in the region of subsequent dehydrogenation. It is postulated that these compounds bind with the carotenoid dehydrogenases, thereby inhibiting carotenogenesis.

The synthesis of a photolabile detergent and its use in the isolation and characterization of protein

Abstract A new ionic detergent sodium 4-(3,3-dimethyl-1-oxotridecyl)benzenesulfonate similar to sodium dodecyl sulfate has been synthesized which is photodegradable to sodium p -acetyl-benzenesulfonate and a simple olefin mixture upon irradiation in aqueous solution with light 300 nm and above in wavelength. This photodegradable detergent can be used to solubilize many proteins and provide information as to the molecular weight and subunit composition of proteins by electrophoresis. The removal of this detergent by photolysis results in no apparent damage to protein.

Purification of geranylgeranyl diphosphate synthase from Phycomyces blakesleanus

Geranylgeranyl diphosphate synthase has been purified to homogeneity from the carotene-overproducing strain M1 of Phycomyces blakesleanus. Usually two activity peaks with molecular weights of 60,000 and 30,000 eluted on gel exclusion chromatography, suggesting that the enzyme consists of two subunits, with a tendency to dissociate. With homogeneous protein, a single-staining band with molecular weight of 30,000 appeared on sodium dodecyl sulfate gel electrophoresis, confirming a subunit molecular weight of 30,000. Only isopentenyl diphosphate and farnesyl diphosphate were accepted by this enzyme for geranylgeranyl diphosphate formation. The smaller allylic compounds, dimethylallyl and geranyl diphosphate, were utilized at less than 1/20th the rate of farnesyl diphosphate. Michaelis constants of 9 microM for isopentenyl diphosphate and 60 microM for farnesyl diphosphate were found. The isoelectric point is 4.8.

Isopentenyl pyrophosphate isomerase:dimethylallyl pyrophosphate isomerase: Isolation from Claviceps sp. SD 58 and comparison to the mammalian enzyme☆

Isopentenyl pyrophosphate isomerase:dimethyl pyrophosphate isomerase (EC 5.3.3.2) has been purified to near homogeneity from Claviceps sp. A molecular weight of 35,000 was found by gel exclusion chromatography as well as by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the enzyme consists of a single subunit and is in contrast to the Mr 22,000 that we have found for the enzyme from liver. The lability of isomerase from liver, often reported, has been found to be due to its susceptibility to proteolysis. Nine compounds have been tested as inhibitors of both isomerases. The binding of analogs requires the pyrophosphate moiety which may be substituted by a variety of alkyl groups. Inclusion of a polar function in the hydrocarbon portion of the analog greatly reduces interaction with the enzyme. Reversibility of the reaction was not found with a higher homolog of the substrate.

The formation of cyclic sesquiterpenes from farnesyl pyrophosphate by prenyltransferase.

Abstract Prenyltransferase, which normally catalyzes the 1′-4 condensation which is the basic polymerization of polyterpenoid and cholesterol biosynthesis, also mediates a pyrophosphate-dependent solvolysis of its substrates. We had previously shown that this was an aberration of the normal reaction and that dimethylallyl alcohol and geraniol were liberated from their pyrophosphate esters (C. D. Poulter and H. C. Rilling, 1976, Biochemistry 15 , 1079–1085) . Enzymatic solvolysis of farnesyl pyrophosphate yielded farnesol, nerolidol, and hydrocarbons. We now demonstrate that the hydrocarbons produced are cyclic Sesquiterpenes such as the germacrenes and the humulenes. We postulate that this results from the structure of the catalytic site which is shaped so that the 9,10 double bond of farnesyl pyrophosphate is forced back over its carbinol carbon. As a result, the carbonium ion which is produced by the enzyme at C-1, by pyrophosphate-induced initiation, can now react with the ω-terminal double bond. This finding strongly supports our proposal for the mechanism for termination of this polymerization reaction catalyzed by prenyltransferase.

Prenylated proteins: demonstration of a thioether linkage to cysteine of proteins.

Prenylated amino acid fragments have been isolated from prenylated proteins of Chinese hamster ovary cells. Gel-exclusion chromatography indicates that these proteins are modified by two different prenyl groups. The prenyl-amino acid fragments are labeled by 35S from cysteine, and this bond is cleaved by Raney-Ni, proving that the prenyl residue is linked to protein via a thioether to cysteine. Hydrazinolysis has been used to demonstrate that the cysteine is carboxy terminal.