Measurement of ligand distribution in individual adsorbent particles using confocal scanning laser microscopy and confocal micro-Raman spectroscopy.

Two methods, confocal scanning laser microscopy and confocal micro-Raman spectroscopy were used to analyse the distribution of IgG antibodies immobilized on CNBr-activated agarose beads. In the first method the internal distribution profile of fluorescent labelled Protein A was used as an indirect measure of the distribution of IgG, while the second method detects vibrations originating from aromatic amino acids present in the immobilized antibodies. Both these methods indicate an homogeneous ligand distribution within IgG Sepharose 4 Fast Flow and IgG Sepharose 6 Fast Flow.

Coupling of 22Na and 36Cl uptake in cultured pigmented ciliary epithelial cells: a proposed role for the isoenzymes of carbonic anhydrase.

Uptake studies with 22Na and 36Cl were performed in cultured bovine pigmented ciliary epithelial cells (PE) to investigate interdependence of Na+ and Cl- transport. (1) 22Na uptake into NaCl depleted cells was stimulated by Cl-. This stimulation was abolished by the simultaneous application of amiloride (1 mM) and bumetanide (0.1 mM), indicating two independent mechanism for Cl- stimulated Na+ uptake: loop diuretic sensitive Na+/Cl- symport and an indirect stimulation of Na+/H+ exchange by Cl-. The latter component of Cl- stimulated Na+ uptake was HCO3- dependent. (2) 36Cl uptake was increased by extracellular Na+. Na+-stimulated Cl- uptake also consisted of two components. One was bumetanide sensitive and the other was blockable by amiloride and partly inhibited by the carbonic anhydrase (CA) inhibitor methazolamide (0.1 mM). (3) Homogenized PE cells were tested for biochemical CA activity using an electrometric method. The cytoplasmic as well as the membrane fraction contained specific CA activity. (4) A model is presented for Na+ and Cl- transport into PE: in addition to Na+/Cl- symport, Na+/H+ and Cl-/HCO3- double exchange may operate in the ciliary epithelium. The latter mechanism provides NaCl uptake into the cell in exchange for H+ and HCO3-, which recycle as CO2 across the membrane. This recycling of CO2 and HCO3-/H+ (and hence indirectly NaCl uptake) is facilitated by the cooperation between membrane bound and cytoplasmic CA.

Renal membrane-bound carbonic anhydrase. Purification and properties.

Microsomes from perfused human donor kidneys were separated by differential centrifugation in sucrose, and thoroughly washed before solubilization by the nonionic detergent nonyl-beta-D-glucoside. The solubilized material was first applied onto an affinity chromatographic column of acetazolamide-oxirane-SepharoseR-CL-4B to remove contaminating cytoplasmic carbonic anhydrase isozymes CA I and CA II. It was then added onto an affinity column of p-aminomethylbenzene sulfonamide coupled to CM Bio-gel AR to purify the membrane-bound carbonic anhydrase activity. This resulted in a 50% pure enzyme. It was then concentrated and fractionated on an anion-exchange column, and desalted and purified to homogeneity (SDS-PAGE and isoelectric focusing) by gel filtration. The enzyme was now purified 411-fold from extractable membrane protein. Its molecular weight was 34.4 kDa from gel filtration and SDS-PAGE, and 36.7 kDa from amino acid analysis. The amino acid composition differed from that of the cytoplasmic isozymes CA I, II, and III. Antisera, produced in rabbits against the purified SDS-treated enzyme, reacted with native nondenatured membrane enzyme protein but only weakly with CA II. Kinetically the enzyme was similar to CA II with respect to hydrase and esterase activities and to inhibition by various sulfonamides. Considered together, the data suggest that the human kidney contains a membrane-bound carbonic anhydrase protein that differs from the cytoplasmic isozymes CA I, II, and III and the secretory form (CA VI) in the saliva.

[Carbonic anhydrase and its function in ion transport in cultivated pigmented ciliary body epithelial cells]. / Die Carboanhydrase und ihre Funktion im Ionentransport in kultivierten pigmentierten Ziliarkörper-Epithelzellen.

The effect of inhibitors of the carbonic anhydrase on aqueous humor formation suggests that carbonic anhydrase has an important role in ion transport across the ciliary epithelium. We therefore investigated the role of carbonic anhydrase in Na+ and Cl- transport in cultured bovine pigmented ciliary epithelial cells (PE) using the radioactive isotopes 22Na and 36Cl. Our findings can be summarized as follows. (1) Na+ uptake into the cell occurs via an amiloride-sensitive Na+/H+ exchanger. (2) Cl- is transported into the cell in exchange for bicarbonate ions. (3) PE cells contain biochemical carbonic anhydrase activity. (4) Na+ uptake into NaCl-depleted cells is markedly stimulated by Cl-. This stimulation is HCO3- dependent and is completely blocked by amiloride and partly inhibited by the carbonic anhydrase inhibitor methazolamide. (5) A model is introduced for transport of NaCl in the ciliary epithelium: carbonic anhydrase is responsible for coupling of Na+/H+ exchange and Cl-/HCO3- countertransport. This model could explain the effect of carbonic anhydrase inhibitors on intraocular pressure.

A radioimmunosorbent technique for the assay of B- and C-types of carbonic anhydrase in human tissues.

A radioimmunosorbent technique for the assay of the human carbonic anhydrase isoenzymes HCA B and HCA C in tissue fluids was developed. The sensitivity of the method was 0.2 ng/ml and the precision was 5% in duplicate determinations for both enzymes. The presence in a tissue of up to 20 times higher concentrations of one isoenzyme will not interfere with the assay of the other. Haemolysates contained (mean +/- SE, n = 11) 12.1 +/- 0.52 and 1.5 +/- 0.06 mg enzyme/g Hb, and serum 0.63 +/- 0.12 and 0.2 +/- 0.02 microgram/ml of HCA B and HCA C, respectively. Pilot experiments indicated that the isoenzymes can be determined also in tissues, i.e. urine, saliva and cerebrospinal fluid, where catalytic methods previously have indicated absence of or only weak carbonic anhydrase activity. N-terminals of both enzymes were not antigenic.