A specific site of vanadium inhibition of cholesterol biosynthesis

PRELIMINARY NOTES

397

A specific site of vanadium inhibition of cholesterol biosynthesis The site of vanadium action in its inhibition of cholesterol biosynthesis has been shown by AZARNOFF AND CURRAN1 to be located between the formation of mevalonic acid and that of squalene. This communication presents data which localize this site of inhibition as squalene synthetase. Male rats of the Holtzman strain weighing 15o-25o g were used in this study. Biosynthetic Iz4Clfarnesyl pyrophosphate was prepared and isolated as described by GOODMANAND P o P J A K 2 except that DL-[2-14Clmevalonic acid was used as substrate instead of [2-z4C~-5-phosphomevalonic acid. The [14C~farnesyl pyrophosphate was incubated with microsomes and a TPNH-regenerating system in a nitrogen atmosphere. Following the incubations, carrier squalene was added and the reaction mixture saponified in ethanolic KOH. The non-saponifiable material (representing primarily radioactive squalene as demonstrated by column chromatography) was extracted 3 times with petroleum ether. The combined petroleum ether extracts were evaporated to dryness in a scintillation vial under a stream of nitrogen. The residue was dissolved in IO ml scintillator (4.0 g diphenyloxazole and o.I g 1,4-bis-2(5-phenyloxazolyl)benzene/1 toluene) and its radioactivity determined in a Packard Tri-Carb Liquid Scintillation Spectrometer. When incubated anaerobically, microsomes supply the squalene synthetase necessary to convert farnesyl pyrophosphate to squalene, but cyclization of the squalene does not occur because of a lack of molecular oxygen. The possibility that the TPNH-regenerating system might be inhibited by vanadium was excluded by assay of glucose 6-phosphate dehydrogenase activity as well as by using T P N H instead of the regenerating system. Thus, from Fig. I it is concluded that vanadium inhibits squalene synthetase. Because the biosynthetic substrate contains a small amount of the other allyl pyrophosphates, 0.2 m M non-radioactive geranyl pyrophosphate 3 was added to the reaction mixture without appreciably influencing the degree of inhibition recorded in Fig. I. This is in agreement with the reported data that microsomes do not appear to convert the other allyl pyrophosphates in the substrate to farnesyl pyrophosphate. .J < ~:

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30

Fig. I. The influence of 4 m M VOSO 4 on the incorporation of [14C]farnesyl p y r o p h o s p h a t e into non-saponifiable (squalene) material. The i n c u b a t i o n m i x t u r e contained: p o t a s s i u m p h o s p h a t e buffer (pH 7.4), o.I M ; MgC12, 5 m M ; T P N , i m M ; glucose 6-phosphate, 2 m M ; N a F , IO m M ; nicotinamide, 3 ° m M ; p l a s m a albumin, 2 m g / m l ; crystalline glucose 6 - p h o s p h a t e dehydrogenase (Boehringer), 2 # g / m l ; microsomes, 3.5 m g p r o t e i n / m l ; [uC]farnesyl p y r o p h o s p h a t e , 72 ooo c o u n t s / min. I - m l incubations were p e r f o r m e d at 37 ° in screw-cap vials vigorously flushed w i t h N,.

Biochim. Biophys. Acta, 51 (1961) 397-398

398

PRELIMINARY NOTES

It is of interest that Mn 2+ can be substituted for Mg ~+ as a cofactor for squalene synthetase 2 since Mn 2+ has been demonstrated 4 capable of overcoming vanadium inhibition of cholesterol biosynthesis in liver clusters. Further studies are in progress to determine if these findings pertain to microsomes themselves. I t has been reported that vanadium lowers coenzyme A levels in intact animals 5 and that it reduces functional ATP levels in crude liver homogenates 6. Whatever the quantitative influence of these factors on cholesterol biosynthesis m a y prove to be, vanadium also specifically inhibits squalene synthetase. This investigation was supported in part by a research grant (H-4882-C2) from the U.S. Public Health Service and (P-26o) from the American Cancer Society.

Department of Medicine, St. Louis University School of Medicine, and the St. Louis University Hospital (Firmin Desloge Hospital), Saint Louis, Mo. (U.S.A.)

DANIEL L.

AZARNOFF

FRANCES ]~. BROCK GEORGE L. CURRAN

1 D. L. AZARNOFF AND G. L. CURRAN, ,]. Am. Chem. Soc., 79 (1957) 2968. 2 DE\V. S. GOODMAN AND G. POPJe{K, J . Lipid Research, I (196o) 286. 3 \V. STOFFEL AND C. MARTIUS, Biochem. Z., 333 (196o) 44 TM 4 G. L. CURRaN, ]. Biol. Chem., 2 ro (1954) 765. 5 E. MASCITELLI-COROIANDOLI AND C. CITTERO, Nature, 183 (1959) 1527. L. D. WRIGHT, L. LI AND R. TRAGER, Biochem. Biophys. Research Communs., 3 (I96o) 264.

Received June Ioth, 1961 Biochim. Biophys. ,4cla, 51 (1961) 397 398

Alternative pathways of enzymic formation of ribotides and ribosides of histamine An established pathway for the enzymic formation of histamine riboside involves interaction of D P N with histamine 1 and subsequent degradation of the resulting histamine dinucleotide by nucleotide pyrophosphatase (to histamine ribotide and adenylic acid) and b y 5'-nucleotidase (to histamine riboside) 2. Confirmatory evidence for the existence of this pathway was recently obtained b y MURAOKA et al2. Triphosphohistamine nucleotide produced b y interaction of T P N with histamine may also be degraded to the ribotide and riboside 4. Preliminary evidence 5 indicates that histamine m a y also interact with 5'-phosphoribose I'-pyrophosphate, leading to the formation of histamine ribotide. We now wish to report evidence for a direct interaction of histamine with either nicotinamide ribotide or nicotinamide riboside, as follows: NMN + + histamine enzyzne___>.5'-ribosylhistamine p h o s p h a t e + nicotinamide + H ÷

(i)

N(ring)-ribosylnicotinamide + histamine enzyme. > N(ring)-ribosylhistamine + nicotinamide + H+

(2)

The enzyme that catalyzes reactions I and 2 is found in abundance in raw bull semen and it probably identical with the pyridine nucleotide nucleosidase first Biochim. Biophys. zJcta, 51 (1961) 398-4oo