Cholesterol synthesis in rat liver peroxisomes. Conversion of mevalonic acid to cholesterol

The key regulatory enzyme of cholesterol, dolichol, and isopentenyl adenosine biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a 97-kilodalton transmembrane glycoprotein which was believed until recently to reside exclusively in the endoplasmic reticulum of mammalian cells. However, several recent publications have shown that the enzyme in liver cells is present not only in the endoplasmic reticulum but also within peroxisomes. In an effort to clarify the role of peroxisomal HMG-CoA reductase, highly purified (95%) rat liver peroxisomes from cholestyramine-treated rats were incubated with RS-[2-14C]mevalonic acid plus cytosolic proteins and then tested for the presence of newly synthesized cholesterol. For comparison, highly purified microsomes from the same liver preparation were incubated at several protein concentrations under the same conditions. A three-step procedure was employed to resolve the newly synthesized cholesterol from the complex mixture of sterol intermediates in cholesterol biosynthesis. After termination of the reaction and addition of a [3H]cholesterol standard, the incubation products were extracted and separated by thin layer chromatography into a number of fractions. The fraction containing C-27 sterols was further resolved by reverse-phase high pressure liquid chromatography. After acetylation, the products were then separated by silicic acid high pressure liquid chromatography. Confirmation of the identity of newly synthesized cholesterol was obtained by recrystallization with added non-radioactive cholestenyl acetate standard. The results indicate that highly purified rat liver peroxisomes are able to convert mevalonic acid to cholesterol in the presence of cytosolic fraction in vitro. An abstract of these results has been published (Krisans, S. K., Thompson, S. L., Burrows, R., and Laub, R. J. (1986) J. Cell Biol. 103, 525 (abstr.).     

The structure of bactoprenol, a lipid formed by lactobacilli from mevalonic acid

1. The name `bactoprenol' has been given to the most abundant lipid formed by three species of lactobacilli from mevalonic acid. 2. A method for the preparation of pure bactoprenol is described. 3. The thin-layer chromatographic properties of bactoprenol and of its acetylated and hydrogenated derivatives resembled those of dolichol. 4. Analysis by mass spectrometry and by nuclear magnetic resonance showed that the molecule is formed by condensation of 10 unsaturated isoprene units and 1 saturated isoprene unit. 5. Its molecular weight is 768 and it has 10 double bonds/molecule. 6. Infrared spectroscopy and the uptake of acetyl groups indicated that the molecule contains a hydroxyl group. 7. It is concluded that bactoprenol is a C₅₅ isoprenoid alcohol.     

Neutrophil-assisted DNA synthesis by human lymphocytes in response to mevalonic acid; enhancement by cytochalasin B

Mevalonic acid is capable of initiating DNA synthesis, morphologic transformation, and cell division in cultures of human peripheral blood lymphocytes. Pure populations of lymphocytes respond poorly to mevalonic acid, but their response can be enhanced by peripheral blood neutrophils. Addition of cytochalasin B (0.5-1.0 micrograms/ml), but not cytochalasin A, to mixed neutrophil-lymphocyte cultures enhances the response of lymphocytes to both Con A and mevalonate, but the increment in mevalonate-induced DNA synthesis (+343%) far exceeds the enhancement which cytochalasin B produces in the Con A response (+24%). As a consequence, the DNA synthetic response in mevalonate (10(-2) M) containing cultures averages 39% of the response to an optimal dose of Con A. The cytochalasin B-enhanced response of mixed neutrophil-lymphocyte cultures to mevalonate is abolished by prior X irradiation of the lymphocytes, or their pretreatment with mitomycin C, as well as by the addition of hydroxyurea to the cultures but is not altered by prior X irradiation or mitomycin C pretreatment of the neutrophil helper population. These experiments suggest that the enhancing effect of cytochalasin B in the response of mixed neutrophil-lymphocyte cultures to mevalonic acid results from its ability to potentiate a time-dependent conditioning effect on lymphocytes which neutrophils exert. The conditioning effect of neutrophils cannot be achieved by cell-free neutrophil lysosomal enzymes released by exocytosis, and reactive oxygen species potentially generated by neutrophils are not involved. Attempts to demonstrate the production by neutrophils of a soluble mediator induced by mevalonate in the presence of cytochalasin B were without success. In the presence of cytochalasin B, only the metabolically active R(-) enantiomeric form of mevalonate is capable of initiating DNA synthesis in mixed neutrophil-lymphocyte cultures. The cytochalasin B-potentiated response of mixed neutrophil-lymphocyte cultures to mevalonic acid is preferentially displayed in cultures containing E-rosette-negative, as opposed to E-rosette-positive, lymphocytes. These observations add to the growing knowledge about the relationship between mevalonate metabolism, DNA synthesis, and cell replication.     

Evidence for modification of lamin B by a product of mevalonic acid

Previous work from this laboratory has shown that a derivative of mevalonic acid is post-translationally incorporated into a number of specific proteins in Swiss 3T3 cells. Neither the nature of the modification nor the identities of the modified proteins have been determined to date. Here we describe results concerning modified proteins of approximately 67 kDa from HeLa cells and Chinese hamster ovary cells. We show that these proteins are specific to the nucleus and remain associated with a Triton/salt-insoluble nuclear fraction. Furthermore, immunological studies demonstrate that one of the modified proteins comigrates on two-dimensional gels with lamin B, a structural protein associated with the nuclear envelope. Using antibodies directed against lamin B in an immunoprecipitation experiment, we further show that this mevalonic acid-modified protein specifically coprecipitates with lamin B. These results support the hypothesis that lamin B is modified by a derivative of mevalonic acid.     

Conversion of calciferols to isotachysterols by trifluoroacetic acid

The conversion of the calciferols to isotachysterols using trifluoroacetic acid is described. The yield of isotachysterol formed is greater by about 10% than that obtained with either antimony trichloride or acetyl chloride. Trifluoroacetic acid also produces a more intense absorption at 500 nm with calciferols than does antimony trichloride, however, the color is less stable.     

Kidney metabolism of mevalonic acid in neonatal chick

• 1.1. Metabolism of mevalonic acid (MVA) to phosphorylated derivatives has been studied in 1–3 day chicks kidneys. In all experiments only 5-phosphomevalonic acid (MVAP) and 5-pyrophosphomevalonic acid (MVAPP) were observed as phosphorylated derivatives of [2-¹⁴C]MVA.
• 2.2. Properties of mevalonate-activating enzymes from neonatal chick kidney have been established. Supplementation of Mg²⁺ or Mn²⁺ strongly increased the MVA phosphorylation. The enzymes require ATP as nucleotide for optimal activity. ITP can also be utilized less effectively.
• 3.3. Addition of 0.1 mM Hg²⁺ or 0..01 mM p-hydorxymerucibenzoate inhibited MVA phosphorylation, thus suggeting that thiol groups may participate in the active site of mevalonate kinase from chick kidney. However, unlike other mevalonate kinases, the enzyme from chick kidney did not require thiol group protectors as activators.
• 4.4. Phosphomevalonae kinase was completely inactivated after 5–10 min treatment at 50°C, whereas mevalonate kinase was found to retain its activity in the same conditions. Exposure to 65°C for 5–15 min resulted in the inactivation of mevalonate kinase.

     

Metabolism of mevalonic acid to long chain fatty alcohols in an insect

A mixture of long chain fatty alcohols (LCFA) with chain lengths of 24 to 30 carbon atoms, as free and esterified components, have been isolated from the aphid, $\text{Schizaphis}\text{graminum}$. Both radiolabelled mevalonic acid and palmitic acid served as effective precursors in the synthesis of LCFA. This is the first time that the $\text{trans}$-methylglutaconate shunt has been shown to be operational for fatty alcohol synthesis and to occur in a lower-evolved animal.     

Conversion of oleic acid to 10-hydroxystearic acid by Nocardia paraffinae

Resting cells of Nocardia paraffinaeCBS 255 58 anaerobically converted oleic acid to 10-hydroxystearic acid (optimum activity at 35°C and pH 7). Addition of oleic acid (0.22 mg/ml of medium), as an inducer, improved the hydroxylating activity. The biocatalyst was inactivated by sonication, lyophilization and heating, but kept 65% of its initial activity after five freeze-thawing treatments. This activity appeared to be located either in the cells or bound to the plasmic membrane.     

Conversion of beta-methylbutyric acid to beta-hydroxy-beta-methylbutyric acid by Galactomyces reessii.

beta-Hydroxy-beta-methylbutyric acid (HMB) has been shown to increase strength and lean mass gains in humans undergoing resistance-exercise training. HMB is currently marketed as a calcium salt of HMB, and thus, environmentally sound and inexpensive methods of manufacture are being sought. This study investigates the microbial conversion of beta-methylbutyric acid (MBA) to HMB by cultures of Galactomyces reessii. Optimal concentrations of MBA were in the range of 5 to 20 g/liter for HMB production. Preliminary shake flask experiments indicated that HMB yields were sensitive to dissolved oxygen levels and that cell growth decreased significantly as MBA concentrations increased. Degradation of HMB was faster at acidic pH, and pH 7.0 was optimal for HMB production. Resting cells obtained from media without MBA could efficiently convert MBA to HMB. Thus, a two-step, fed-batch fermentation procedure in which biomass was first produced, followed by coaddition of MBA and glucose, while dissolved oxygen was maintained at 20% of saturation, was designed. A maximum HMB concentration of 38 g/liter was obtained after 136 h, and the molar conversion yield was more than 0.50 mol of HMB/mol of MBA during the fermentation.