Benjamin Frydman

Properties of purified recombinant human polyamine oxidase, PAOh1/SMO.

The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.

Induction of the PAOh1/SMO polyamine oxidase by polyamine analogues in human lung carcinoma cells

PurposeThe induction of polyamine catabolism has been directly associated with the cytotoxic response of various tumor types to the antitumor polyamine analogues. Initially, human polyamine catabolism was assumed to be under the control of a rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (PAO). We have recently cloned a new polyamine analogue-inducible human polyamine oxidase (PAOh1/SMO) that efficiently uses spermine as a substrate. The induction of PAOh1/SMO in response to multiple polyamine analogues was examined in representative lung tumor cell lines.MethodsRepresentatives of three different classes of antitumor polyamine analogues were examined for their ability to induce PAOh1/SMO.ResultsThe human adenocarcinoma line, NCI A549 was found to be the most responsive line with respect to induction of PAOh1/SMO in response to analogue exposure. Similar to previous observations with SSAT expression, PAOh1/SMO induction was found to occur primarily in non-small-cell lung cancers cell lines. Using a series of polyamine analogues, it was found that the most potent inducers of PAOh1/SMO possessed multiple three-carbon linkers between nitrogens, as typified by N1,N11-bis(ethyl)norspermine.ConclusionsSince PAOh1/SMO is an analogue-inducible enzyme that produces H2O2 as a metabolic product, it may play a significant role in determining the sensitivity of various human tumors to specific polyamine analogues.

Antizyme induction by polyamine analogues as a factor of cell growth inhibition

The polyamines spermidine and spermine and their diamine precursor putrescine are essential for mammalian cell growth and viability, and strategies are sought for reducing polyamine levels in order to inhibit cancer growth. Several structural analogues of the polyamines have been found to decrease natural polyamine levels and inhibit cell growth, probably by stimulating normal feedback mechanisms. In the present study, a large selection of spermine analogues has been tested for their effectiveness in inducing the production of antizyme, a key protein in feedback inhibition of putrescine synthesis and cellular polyamine uptake. Bisethylnorspermine, bisethylhomospermine, 1,19-bis-(ethylamino)-5,10,15-triazanonadecane, longer oligoamine constructs and many conformationally constrained analogues of these compounds were found to stimulate antizyme synthesis to different levels in rat liver HTC cells, with some producing far more antizyme than the natural polyamine spermine. Uptake of the tested compounds was found to be dependent on, and limited by, the polyamine transport system, for which all these have approximately equal affinity. These analogues differed in their ability to inhibit HTC cell growth during 3 days of exposure, and this ability correlated with their antizyme-inducing potential. This is the first direct evidence that antizyme is induced by several polyamine analogues. Selection of analogues with this potential may be an effective strategy for maximizing polyamine deprivation and growth inhibition.

A preparative synthesis of lapachol and related naphthoquinones

Abstract The lithium salt of 2-hydroxy-1,4-naphthoquinone was prepared in situ by addition of lithium hydride to the frozen solution of the quinone in dimethyl sulfoxide. As the solution thawed, the lithium quinone was slowly formed and was then alkylated with 3,3-dimethylallyl bromide. Lapachol was thus obtained in 40% yield. When treated with m-chloroperoxybenzoic acid it was converted into its epoxide, that was cyclized with boron trifluoride etherate to 3-hydroxy-β-lapachone in 67% overall yield. Esters of the latter were prepared by condensation with carboxylic acid derivatives using 1,1′-carbonyldiimidazole and DBU as condensing agents.

Concerning the specificity of heme oxygenase: The enzymatic oxidation of synthetic hemins

Abstract Hemin XIII 4 ∼ , hemin III 5 ∼ , and iron 1,4-di(β-hydroxyethyl)porphyrin 6 ∼ were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. These are all better substrates of the oxygenase than the natural substrate, hemin IX 1 ∼ . The enzymatic oxidation was selective for the α-methine bridge and in every case only the α-biliverdins were obtained. The latter were readily reduced by biliverdin reductase to the corresponding α-bilirubins. The absence of isomers in addition to the α-bilirubins was established by preparing the derived azopigments and by using [α-14C] 6 ∼ and [α-14C] 4 ∼ as substrates. The chemical oxidation of 4 ∼ , 5 ∼ , and 6 ∼ gave the expected mixture of biliverdins. It is concluded that heme oxygenase is not specific for hemin IX. On the other hand, the enzyme is highly selective for the α-methine bridge, defined as the methine opposed to that flanked by the 6,7-propionic acid residues.

Purification and properties of porphobilinogen deaminase from wheat germ

Abstract Porphobilinogen deaminase from wheat germ was purified about 1000-fold. A series of DEAE-cellulose fractionations gave varying yields of uroporphyrin formation. The properties of the purified wheat germ enzyme were studied and compared with PBG deaminases from other origins such as Swiss chard leaves, human erythrocytes, and Rhodospirillum rubrum . A series of inhibitory effects on the enzyme were examined and the results are discussed in terms of enzyme structure and reactivity. The substrate specificity of the enzyme toward several 2-methylamino pyrroles was examined. A dithionite-activated protein factor was also isolated from wheat germ and Swiss chard and was found to consume PBG in a special way and to interact with the PBG-deaminase by inhibiting its activity. An hypothesis of the mode of action of deaminase is discussed in light of the data presented.

Antizyme induction mediates feedback limitation of the incorporation of specific polyamine analogues in tissue culture.

Spermidine, spermine and putrescine are essential for mammalian cell growth, and there has been a pervasive effort to synthesize analogues of these polyamines that will disrupt their function and serve as tools to inhibit cell proliferation. Recently, we demonstrated that a number of such polyamine analogues are also capable of inducing the regulatory protein AZ (antizyme). In the present study the incorporation of a few sample analogues [mimics of bis(ethyl)spermine] was shown to be significantly limited by a decrease in the V(max) for the polyamine transport system in response to analogue-induced AZ. This creates an unusual circumstance in which compounds that are being designed for therapeutic use actually inhibit their own incorporation into targeted cells. To explore the impact of this feedback system, cultures of rat hepatoma HTC cells were pre-treated to exhibit either low or high polyamine uptake activity and then exposed to polyamine analogues. As predicted, regardless of initial uptake activity, all cultures eventually achieved the same steady-state levels of the cellular analogue and AZ. Importantly, analogue-induced AZ levels remained elevated with respect to controls even after the native polyamines were reduced by more than 70%. To model the insufficient AZ expression found in certain tumours, GS-CHO (GS Chinese-hamster ovary) cells were transfected to express high levels of exogenic AZI (AZ inhibitor). As anticipated, this clone incorporated significantly higher levels of the polyamine analogues examined. This study reveals a potential limitation in the use of polyamine-based compounds as therapeutics, and strategies are presented to either circumvent or exploit this elegant transport feedback system.

Pyrrolooxygenases: Isolation, properties, and products formed

Abstract 1. 1.|A new group of enzymes was isolated from wheat germ and rat liver which oxidized the pyrrole ring of indoles affording o -formamidophenacyl derivatives. They behaved as mixed-function oxidases and were named pyrrolooxygenases. Tryptophan, ethyl N -acetyltryptophan, skatole, 3-indoleacetic acid, 3-indolepropionic acid and indole were substrates of the pyrrolooxygenases. The enzymatic oxidations were catalyzed by at least two enzymes within the group; one acting on tryptophan and its derivatives, and the other one acting on skatole and the other indoles. 2. 2.|The pyrrolooxygenases had an absolute requirement for oxygen and an exogenous reducing agent. The reducing agents were illuminated chloroplasts for the plant enzymes, and NADPH and a microsomal transport system for the mammalian enzymes. Both could be replaced by sodium dithionite. 3. 3.|Chelating agents, such as α,α′-dipyridyl and EDTA inhibited the enzymatic activity, while sodium cyanide had no effect. The enzymes were also inactivated by dithiothreitol and mercaptoethanol. 4. 4.|A natural heat-labile macromolecular inhibitor of the pyrrolooxygenases was present in the crude extracts and was separated during the successive purification steps. 5. 5.|Formamidase (EC 3.5.1.9) activity was present in the extracts together with the pyrrolooxygenases and transformed the o -formamidophenacyl derivatives into the corresponding o -amino derivatives. During the purification steps the formylase activity was partly removed.

Interconversion of the molecular forms of biliverdin reductase from rat liver.

Rat liver biliverdin reductase exists in two molecular forms. The major molecular form 1 has a high reduction rate for biliverdin IX alpha, while the minor molecular from 2 has high reduction rates for both biliverdins IX alpha and IX beta. The major molecular form 1 was gradually transformed into a second major form (form 3) by treatment of the rats with CoCl2. Form 1 reduces biliverdin IX alpha at two and a half times the rate of biliverdin IX beta while form 3 reduces both isomers at about the same rate. This transformation involves a de novo mRNA and protein synthesis since it could be prevented by cycloheximide and actinomycin D. Molecular form 3 can be transformed back into molecular form 1 by an in vitro treatment with reduced thioredoxin. Phenylhydrazine treatment also induced the transformation of molecular form 1 into molecular form 3 in rat liver. Biliverdin reductase from rat spleen and kidney exists only in molecular form 1 and is not transformed into molecular form 3 by either CoCl2 or phenylhydrazine treatments.

Isolation and properties of wheat germ uroporphyrinogen III cosynthetase

Abstract 1. 1. A reproducible method for the isolation of uroporphyrinogen III cosynthetase from wheat germ is described. 2. 2. Some properties of the isolated enzyme were studied. The pH optimum was 8.2. The enzyme was very sensitive to heat and was inhibited by several salts.