Tokyo Biochemical Research Foundation

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 microM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3'-phosphoadenosine 5'-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.

Spermidine synthase was purified to homogeneity from rat and pig liver by a method modified from a previously reported one using DEAE-Sepharose, S-adenosyl(5')-3-thiopropylamine-Sepharose affinity chromatography, Sephacryl S-300 gel filtration and polyacrylamide gel electrophoresis. No apparent difference between the two enzymes was observed in specific activity, molecular weight (74,000), or subunit composition (two subunits). However, significant differences were observed in their pI values, which were 5.16 for the pig enzyme and 5.34 for the rat enzyme, and their peptide maps. Amino acid compositions of the two enzymes were closely related, but differed significantly in some amino acids. In addition, the rat enzyme was more sensitive to inhibition by S-adenosyl-1,8-diamino-3-thiooctane than the pig enzyme.

Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5')-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5'-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.

The pK values of thermospermine, a novel asymmetric tetraamine (4,8-diazadodecane-1,12-diamine), have been determined for the first time by the conventional titration method with subsequent data analyses. It was found to be the most basic polyamine among naturally occurring tetraamines. The 15N NMR titration method was applied to determine its protonation site. Natural-abundance 15N magnetic resonance spectra of the polyamine have been recorded as a function of pH from its free to the protonated form. Its apparent pK values, as well as the intrinsic chemical shifts for each stage of protonation, have been calculated from the NMR titration curve by using a nonlinear least-squares method. The four protonation sites of thermospermine have been successfully determined to be N12, N1, N-8, and N-4 in that order from the free to the completely protonated form. In a similar manner the protonation sites of spermidine and two symmetric tetraamines, thermine and spermine, have been established. 13C NMR experiments for the four polyamines were almost consistent with those of 15N NMR. Owing to the much higher concentration of the polyamines furnished for NMR titration experiments, the pK values obtained by NMR were larger than those derived by the conventional titration methods.

The distribution of enzymes involved in polyamine synthesis and that of the polyamine levels were investigated in the duodenal mucosa of vitamin D-deficient chicks and those supplemented with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. Duodenal epithelial cells were isolated sequentially from the villus tip to the crypt region using a nonenzymatic method. In vitamin D-deficient chicks, the activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase were markedly higher in the crypt cells than in the villus cells. Similar gradients were observed in the distribution of putrescine and spermidine. Six hours after iv administration of 625 ng 1 alpha,25-(OH)2D3, ODC activity and putrescine levels markedly increased both in the villus and crypt region. The rate of the increase in ODC activity and putrescine levels, however, was much higher in the villus than in the crypt region. Neither S-adenosylmethionine decarboxylase activity nor spermidine levels were affected by the treatment with 1 alpha,25-(OH)2D3. To investigate differential uptake of 1 alpha,25-(OH)2D3 in the duodenal mucosa, 0.1 nmol 1 alpha,25-(OH)2[3H]D3 with or without a 200-fold excess of unlabeled 1 alpha,25-(OH)2D3 was injected into rachitic chicks, and epithelial cells were sequentially isolated 2 h later. The radioactivity specifically incorporated in the nuclear fraction was distributed uniformly from the villus tip to crypt cells. The cytoplasmic receptor for 1 alpha,25-(OH)2D3 was similarly distributed in the crypt and villus cells. These results suggest that 1 alpha,25-(OH)2D3 plays a physiological role in cellular activities not only in the villus but also in the crypt region through polyamine biosynthesis.

1. The metabolic fates of N-n-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and N,N-di-n-butylnitrosamine (DBN) were investigated in the rat and other animal species, to elucidate a possible relationship between metabolism and organotropic carcinogenicity to the urinary bladder of these N-nitrosamines. 2. The principal urinary metabolite of BBN as well as of DBN in the rat was N-n-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), which was demonstrated to be the active form of these compounds as bladder carcinogen. The species difference in response to BBN or DBN is discussed on the basis of the urinary excretion rate of BCPN. 3. Metabolism in vivo and carcinogenicity of a number of BBN analogues were investigated in the rat and a general scheme for biotransformation of N-alkyl-N-(omega-hydroxyalkyl)nitrosamines is given. 4. A possible correlation of structure and metabolism with organotropic carcinogenicity of BBN analogues is discussed, with special reference to selective induction of bladder tumours. 5. A clear demonstration of overlapping carcinogenic and mutagenic activities is presented for BBN, DBN and related compounds.

3-Oxo-12β, 14-dihydroxy-14β-carda-4, 20(22)-dienolide (VII) derivable from digoxigenin (V) was treated with lithium borohydride in pyridine to give 5α-digoxigenin (VIa), which was demonstrated to be identical with the natural cardenolide aglycone, syriogenin. The structure of syriogenin was thus firmly established as 3β, 12β, 14-trihydroxy-5α, 14β-card-20(22)-enolide.

Abstract Beim Erhitzen des 3‐Oxodigitoxigenins (I) mit einem Palladiumkohle‐Katalysator in siedendem Triglyme bildet sich ein Gemisch der Verbindungen (II)‐(V), die sich nur schwer voneinander trennen lassen.

Serum D-glucaric acid (V) level was determined by the colorimetric method, especially after oral administration of D-glucuronolactone (I) or D-glucarolactones (II, III) to man. The highest level was observed at one hour after administration simultaneously with the lowest level of serum β-glucuronidase activity as well as the maximal β-glucuronidase inhibition of serum. In vivo conversion of I into β-glucuronidase inhibitor was thus demonstrated.