Photoaffinity labeling with underivatized T3 was used to identify T3-binding proteins in the membrane of rat erythrocytes. UV irradiation of ghosts and peripheral protein-depleted membranes in the presence of [125I]T3 resulted in the covalent attachment of 125I to membrane proteins (analyzed by polyacrylamide gel electrophoresis and autoradiography). In the presence of the free radical scavenger dithiothreitol, 125I was selectively incorporated into a 45,000 mol wt band (p45) that was an integral membrane polypeptide. p45 photolabeling was half-inhibited by 14 nM unlabeled T3. This concentration is similar to the Km for T3 transport in rat erythrocytes and the Kd of the high affinity T3-binding sites under equilibrium binding conditions in the rat erythrocyte membrane. T4 and tryptophan also strongly inhibited p45 labeling, whereas the D-isomer of T3 was less efficient, and leucine had no effect. This corresponds to the specificity of the system T-related T3 transport system and T3-binding sites of rat erythrocytes. The SH-reagent N-ethylmaleimide prevented p45 labeling, unless T3 was present to protect the T3 transport activity and the high affinity T3-binding sites from inactivation. No saturable labeling of p45 or other polypeptides was detected in membranes prepared from human erythrocytes, which have very low T3 transport activity and no measurable high affinity T3-binding sites. p45 is not disulfide linked and is not a degradation product of higher mol wt polypeptides. Substrates and specific inhibitors of known erythrocyte membrane transporters did not alter p45 photolabeling, indicating that p45 is not functionally related to these transporters. We conclude that the photoaffinity-labeled T3-binding protein p45 has the properties expected of the T3-binding component of the T3 transport system in rat erythrocytes.

The transport of L-T3 was studied in washed rat erythrocytes. L-T3 uptake was temperature sensitive: the initial velocity of uptake at low substrate concentration was 40 times higher at 37 C than at 0C whereas, at equilibrium, the ratio of cell-associated to extracellular L-T3 was about 7 times lower at 37 C than at 0 C. When [125I]L-T3-loaded erythrocytes were diluted into a serum albumin-containing medium, the efflux of L-T3 proceeded at a rate similar to that of influx. A large excess of unlabeled L-T3 in the medium blocked influx and efflux of labeled L-T3, indicating a saturable carrier-mediated transport process across the plasma membrane. the transport obeyed simple Michaelis-Menten kinetics with an apparent Km of 53 nM and a Vmax of 4.3 pmol/min.10(8) cells at 0 C. The Km increased only slightly with temperature whereas the Vmax was 100 times higher at 37 than at 0 C. The Arrhenius activation energy of uptake was 21 Cal/mol. The nonsaturable adsorption of L-T3 to the cells did not exceed 1% of the equilibrium levels at 0 C and 10% at 37 C. Uptake of L-T3 was very specific: unlabeled L-T4, D-T3, triiodothyroacetic acid, rT3, and DL-thyronine inhibited uptake with inhibition constant (Ki) values which were 35, 60, 65, 110, and 250 times, respectively, greater than the Km of L-T3. [125I]L-T4 uptake was negligible. L-T3 uptake and L-T4 inhibition of L-T3 uptake were pH dependent. It is suggested that only the unionized 4'-OH forms of the hormones were recognized by the transport system. At equilibrium, L-T3 was accumulated within the cell (apparent intracellular concentration approximately 50 times higher than that in the medium at 37 C). However, uptake was not dependent on the transmembrane Na+ gradient, suggesting facilitated rather than active transport. Analysis of L-T3 binding to erythrocyte cytosolic proteins suggested that they were implicated in the intracellular trapping of L-T3. At a concentration of 5 x 10(9) erythrocytes/ml (approximately the blood concentration), the amount of L-T3 accumulated in the cells was 13.5 times higher than the extracellular amount. We conclude that L-T3 is solely transported by a saturable, stereospecific, and Na+-independent carrier system. The intracellular accumulation and the rapid transmembrane movements of L-T3 suggest that erythrocytes might play a role in the interorgan transport of L-T3.

No abstract

Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 X 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at -20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.

The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding. THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides. Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo mu-crystallin. This protein is a major structural kangaroo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Purified GST fusion protein, but not GST, bound T3 specifically with high affinity [dissociation constant (Kd) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [(125)I]T3. T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH [activation constant (K[act]) = 10(-8) M], but not by NADH. The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.