Isolation and partial characterization of a vitamin K-dependent carboxylase from bovine aortae.

Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu ('FLEEL'), decarboxylated osteocalcin, decarboxylated fragment 13-29 from descarboxyprothrombin and decarboxylated sperm 4-carboxyglutamic acid-containing (Gla-)protein. The results obtained demonstrated that liver and vessel wall carboxylase may be regarded as isoenzymes with different substrate specificities. The newly discovered enzyme is the first vitamin K-dependent carboxylase which shows an absolute substrate specificity: FLEEL and decarboxylated osteocalcin were good substrates for vessel wall carboxylase, but decarboxylated fragment 13-29 and decarboxylated sperm Gla-protein were not carboxylated at all.     

Bone markers during a 6-month space flight: effects of vitamin K supplementation

Rapid bone loss is a serious health problem for astronauts during long lasting missions in space. We have recorded the changes of biochemical markers for bone metabolism in one of the astronauts during the 6-month space flight of the EUROMIR-95 mission. Immediately after launch both bone resorption markers and urinary calcium excretion increased about two fold, whereas bone formation markers remained unchanged. After 12 1/2 weeks the astronaut received vitamin K1 (10 mg/day for 6 weeks). Vitamin K is known to be involved in the formation of gamma-carboxyglutamate (Gla) in proteins, such as the calcium-binding bone Gla-proteins osteocalcin and matrix Gla-protein. Concomitant with the start of vitamin K treatment, the calcium-binding capacity of osteocalcin increased, and so did the urinary excretion of free Gla. This is suggestive for a subclinical vitamin K-deficiency in the astronaut before vitamin K-supplementation. During periods of high vitamin K status markers for bone formation (osteocalcin and bone alkaline phosphatase) had increased as compared to the first part of the flight. The mean increases were 14 and 23%, respectively. Our data suggest that increased intake of vitamin K may contribute to counteracting microgravity-induced loss of bone mass during long lasting space missions, but need confirmation in more astronauts.     

The rhizosphere in Zea: new insight into its structure and development

Some of the nodal roots of field-grown Zea mays L. bear a persistent soil sheath along their entire length underground except for a glistening white soil-free zone which extends approximately 25 mm behind the root cap. These roots are generally unbranched. The histology of the surface and the rhizosphere of the sheathed roots has been examined by correlated light and electron microscopy. All mature peripheral tissues including root hairs, are largely intact and apparently alive where enclosed by the soil sheath. The sheath is permeated by extracellular mucilage which is histochemically distinct from the mucilage at the epidermal surface, but similar to that produced by the root cap. Isolated cells resembling those sloughed from the sides of the root cap persist in the soil sheath along the length of these roots. Fresh whole mounts of the sheath show that these detached cells may be alive and streaming vigorously even at some distance from the root cap. Rhizosphere mucilage is associated with the isolated cells.To whom correspondence should be addressed     

The formation of thromboxane B2, leukotriene B4 and 12-hydroxyeicosatetraenoic acid by alveolar macrophages after activation during tumor growth in the rat

Pieces of tumor tissue were implanted subcutaneously in the right flank of BN female rats. After 3, 7, 10, 12, 14 and 17 days the lungs were lavaged and the alveolar macrophages collected. The cells were activated with the calcium ionophore A23187 and the formation of thromboxane B2 (TxB2), leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid (12-HETE) determined. The formation of TxB2 decreased considerably until day 7. Thereafter, no changes occurred. The formation of LTB4 increased after the tumor implantation until day 10 and remained stable for the rest of the period, 12-HETE formation was approximately similar, with a decrease at day 12 but continued to increase after day 14. These results suggest that during tumor growth an inhibition of the cyclo-oxygenase or thromboxane synthase occurs and an activation of the C5- and C12-lipoxygenases of the alveolar macrophages.     

Purification and properties of two lipases from pig adipose tissue.

Two lipases were purified from pig adipose tissue after delipidation by a mild and effective procedure using mixtures of chloroform and butanol. This was followed by hydrophobic adsorption chromatography on aminohexyl-Sepharose 4B coupled with octanoic acid, gel filtration on Sephadex G-100, and isoelectric focusing. Two electrophoretically and chromatographically pure enzymes were obtained, which had the same molecular weight (60 000 +/- 3000) and specific activity, and almost identical amino acid compositions; the isoelectric points, i.e. 5.2 and 5.5, differed.     

Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.     

Isoenzymes of vitamin-K-dependent carboxylase

Vitamin-K-dependent carboxylase was prepared from bovine liver, kidney, lung and testis and it was checked that these systems obeyed the laws of normal enzyme kinetics. Four carboxylatable substrates were obtained from different sources and the apparent Michaelis constants of the various carboxylases for these four substrates were measured. From the results thus obtained we concluded that carboxylase is a group name for a number of isoenzymes which are present in hepatic as well as in various non-hepatic tissues.