Hi Jon,

With all due respect to your approach to isolate microsomes from cultured LNCaP cells, I think it takes a village to obtain enough amounts of ER from this object, as the only fraction I’ve known about was so called “crude microsomes” from cultured LNCaP cells. Instead, I’m almost sure you might measure various UGT activities in intact cultured LNCaP cells, i.e. without problematic and laborious isolation of microsomes. Why am I sure? Just because I am familiar with the excellent enzyme-kinetic paper (and some its authors) on the very subject published 20 years ago. Beneath please find an abstract (truncated) and the paper publication information source.

Recent findings obtained by our group showed that incubation of LNCaP cells with labeled steroids leads to the formation of 3- and 17-hydroxysteroid glucuronides. Results indicate that the UGT has a high affinity for testosterone, dihydrotestosterone, androsterone and androstane-3 alpha, 17 beta-diol (3 alpha-DIOL), with Km values ranging from 0.25 to 0.68 microM. The relative specificities (Vmax/Km) also showed higher turnover rates for testosterone, DHT, ADT and 3 alpha-DIOL. Dixon plot and Cornish-Bowden analysis demonstrate that testosterone, DHT, ADT, and 3 alpha-DIOL inhibit the glucuronidation of DHT and ADT in a competitive fashion. Further evidence for the presence of two UGTs in LNCaP cells was obtained by incubation with a variety of 3 beta-OH-C19 steroids which caused a marked inhibition of DHT-G formation but had no effect on the glucuronidation of ADT. In summary, our data demonstrate the presence of at least two UGTs in the human prostate cancer cell line LNCaP.

Guillemette C1, Hum DW, Bélanger A. Specificity of glucuronosyltransferase activity in the human cancer cell line LNCaP, evidence for the presence of at least two glucuronosyltransferase enzymes. J Steroid Biochem Mol Biol. 55(3-4):355-62, 1995.

Best wishes,

Ilya

Hi, Jon,

If you still eager to have a protocol for isolating (crude) microsomes from cultured LNCaP cells, I would refer you to the paper of White et al in Genome Biol. 2004; 5(1): R4, entitled “Identification of androgen-coregulated protein networks from the microsomes of human prostate cancer cells. They, in turn, used to use the protocol described by Han et al. in Nat Biotechnol. 2001;19:946–951. Namely, cells were grown in RPMI medium supplemented with 10% FBS to a density of 1 × 106 cells/ml, and microsomal fractions were prepared as following. Equal numbers of cells (1 × 108) were either mock-treated or treated with 10 nM PMA for 1 h at 37°C. Cells were then washed with ice-cold PBS, swelled for 20 min, and lysed in a hypotonic buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 mM CaCl2) supplemented with protease inhibitor cocktail (Boehringer Mannheim). Cells were then spun for 15 min at 3,000 g to remove nuclei and cytoskeletal components. The supernatant was further spun for 2 h at 100,000 g to pellet the microsomal fraction. The microsomal pellet contained plasma membrane, Golgi, endoplasmic reticulum, mitochondria, lysosomes, and all other membrane-bound vesicles separated from soluble cytosol. Western analysis of known membrane, mitochondria-associated proteins, and known cytosolic proteins in identically prepared subcellular fractions from human lymphocytes indicated at least 20-fold enrichment of membrane proteins in the microsomal fraction compared to total cell lysates. Resulting pellets were dissolved by boiling in labeling buffer (50 mM Tris-HCl pH 8.3, 5 mM EDTA, 0.5% SDS), and the protein concentration was measured.

Best wishes,

Ilya