Abstract
The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes. As obtained from vitamin K-deficient rat liver, this soluble preparation is dependent upon the in vitro addition of vitamin K1 for carboxylating activity. The enzyme system is complex and is dependent upon NADH and dithiothreitol for maximum activity. While detergents used to solubilize the enzyme complex do markedly inhibit the activity of the system, the solubilized system is still highly responsive to vitamin K addition and can be used for further study of the carboxylating enzyme system. The requirement for dithiothreitol and the inhibition by p-hydroxymercuribenzoate indicate the involvement of an --SH enzyme in the carboxylating system.
Highlights
From the Vitamin and Nutrition Research Laboratory, Oklahoma Medical Department of Biochemistry and Molecular Biology, College of Medicine, Health Sciences Center, Oklahoma City, Oklahoma 73104
The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes
The percentages of the activity released by Lubrol PX and by Triton X-100 are given in Table V, indicating approximately 40% release into the supernatant of the enzyme system present in the detergent-treated microsomes
Summary
Preparation of Animals-Vitamin K-deficient rats are prepared by feeding the vitamin K-deficient diet of Mameesh and Johnson [11] to 175- to 200-g male Sprague-Dawley rats, housed in coprophagypreventing cages [12]. This microsomal pellet was resuspended in 0.5 volume of homogenizing buffer. The microsomal pellet was resuspended in % volume of the homogenizing somal protein/ml. 1 rn~ dithiothreitol, as noted under “Results.” This microsomal suspension was shaken for 10 min at mom temperature and centrifuged for 1 hour at 105,000 x g to remove any insoluble material. Incubations were done on l-ml samples of the detergent-treated microsomes and the 105,000 x g supernatant after addition of 0.2 ml of the ATP-generating . This rabbit antiserum to rat prothrombin was used to further identify the CarhoxylLlabeled prothrombin formed during in vitro incubations and to identifv the carboxyl-labeled prothrombin formed in uiuo following administration of vitamin K, and I’“C lbicarbonate to vitamin K-deficient rats.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
