A dual-monoclonal sandwich assay for prostate-specific membrane antigen: levels in tissues, seminal fluid and urine.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a 750-residue integral membrane glycoprotein and the target of an in-vivo imaging agent for metastatic prostate carcinoma (PCa). PSMA expression in normal and diseased prostatic tissues has previously been demonstrated by immunohistochemical techniques. In order to quantify PSMA levels in tissue homogenates and physiological fluids, we have developed a dual monoclonal antibody (mAb) sandwich assay which detects the antigen at a sensitivity <1 ng/mL and which is linear across the working range 0-50 ng/mL. METHODS: The assay involves capture of the PSMA by a biotinylated mAb (7E11) immobilized onto a streptavidin-coated microtiter plate; this mAb binds to the N-terminus of the antigen. The captured PSMA is detected by an Eu-labelled mAb (PEQ226) which binds in the region corresponding to Residues 134-437 of the antigen. PSMA was purified from LNCaP cells by immunoaffinity chromatography, and used as a calibrator, based on its concentration by the bicinchoninic acid (BCA) protein assay. RESULTS: The assay was applied to a panel of normal and tumor tissues. Levels were highest in the prostate tissues (292-4254 ng/mg protein). Low levels (21-51 ng/mL) were observed in membranes from ovary and breast, and neglible levels (1-10 ng/mg) in membranes from skin, liver, intestine, and kidney. Levels in the corresponding cytosol fractions were 20-to 50-fold lower. The average PSMA level in seminal fluid from 21 donors was 9, 012 ng/mL. On average, levels in normal-male urine (3.47 ng/mL) were ten-fold higher than in normal-female urine (0.3 ng/mL). CONCLUSIONS: This report is the first to describe absolute quantitation of PSMA in tissues and fluids. Congruent with earlier tissue studies based on immunohistochemical staining and Western-blot analysis, prostate tissue membranes expressed the highest levels of PSMA.

Studies on the transverse tubule membrane Mg-ATPase. Lectin-induced alterations of kinetic behavior.

Transverse tubule (TT) membrane vesicles contain a very active Mg-ATPase (EC 3.6.1.3). Concanavalin A (ConA) and other lectins were found to activate the TT Mg-ATPase from chicken skeletal muscle up to 25-fold yielding specific activities greater than 800 mumol/h/mg. The sarcoplasmic reticulum Ca-ATPase and the sarcolemma Na,K-ATPase were unaffected by ConA. 125I-Labeled lectin binding to the TT membrane Mr 102,000 glycoprotein supports the contention that this protein is identical with or is intimately associated with the TT Mg-ATPase. The ATPase exhibited non-Michaelis-Menton kinetics with both apparent negative cooperativity (n = 0.723; S0.5, Mg-ATP = 14 microM) and substrate inhibition (Ki, Mg-ATP = 10.2 mM), both of which were eliminated in the presence of ConA. Under the same conditions, ConA also abolished the unusual temperature dependence and potent Triton X-100 inhibition. The similarities in ConA suppression of both Triton and substrate inhibition suggest that these ligands may be interacting through a non-catalytic site and that Triton is serving as a nucleotide-mimetic agent. The unique kinetic responses are consistent with a homotropic substrate modifier mechanism wherein the enzyme can be viewed as possessing a single catalytic and a single regulatory site on a single polypeptide chain. It is proposed that ConA interferes either with ligand interaction at a putative regulatory site or blocks communication between a regulatory site and the catalytic site. The possible nature of the regulatory site and its modulation by a ConA-like, endogenous, skeletal muscle lectin and their combined role in excitation-contraction coupling is discussed.

Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells.

Human umbilical vein endothelial cells incorporate eicosapentaenoic acid (EPA) when this fatty acid is present in the culture medium. From 30 to 70% of the uptake remains as EPA, and much of the remainder is elongated to docosapentaenoic acid. All of the cellular glycerophospholipids become enriched with EPA and docosapentaenoic acid, with the largest increase in EPA occurring in the choline glycerophospholipids. When this fraction is enriched with EPA, it exhibits a large decrease in arachidonic acid content. Cultures exposed to tracer amounts of [1-14C]linolenic acid in 5% fetal bovine serum convert as much as 17% of the radioactivity to EPA. The conversion is reduced, however, in the presence of either 20% fetal bovine serum or 50 microM linolenic acid. Like arachidonic acid, some newly incorporated EPA was released from the endothelial cells when the cultures were exposed to thrombin. However, as compared with arachidonic acid, only very small amounts of EPA were converted to prostaglandins. Cultures enriched with EPA exhibited a 50 to 90% reduction in capacity to release prostacyclin (PGI2) when subsequently stimulated with thrombin, calcium ionophore A23187, or arachidonic acid. The degree of inhibition was dependent on the time of exposure to EPA and the EPA concentration, and it was not prevented by adding a reversible cyclooxygenase inhibitor, ibuprofen, during EPA supplementation. EPA appears to decrease the capacity of the endothelial cells to produce PGI2 in two ways: by reducing the arachidonic acid content of the cell phospholipid precursor pools and by acting as an inhibitor of prostaglandin production. These findings suggest that regimens designed to reduce platelet aggregation and thrombosis by EPA enrichment may also reduce the capacity of the endothelium to produce PGI2.

A rapid, isocratic method for phospholipid separation by high-performance liquid chromatography.

A rapid, isocratic method for separating the most prevalent phospholipids by high-performance liquid chromatography is described. Baseline resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin is achieved in less than 40 min on a silica column. Lipids are injected in 10 microliter of chloroform-diethyl ether 1:2 (v/v) and eluted with a solvent mixture of acetonitrile-methanol-sulfuric acid 100:3:0.05 (v/v/v) at a flow rate of 1 ml/min. Neutral lipids and cardiolipin elute with the solvent front. Chromatography of a radioactive cell lipid extract indicates a recovery of better than 97%. The procedure is sensitive enough to permit the analysis of the main phospholipids present in a monolayer culture containing about 100 micrograms of cell protein.