A cDNA clone encoding a human cytosolic thyroid hormone-binding protein (p58) has been isolated. The human sequence was found to be homologous to that of rat pyruvate kinase (EC 2.7.1.40) subtype M2. p58 is a monomer that has --5% the enzymatic activity of the tetrameric pyruvate kinase M2. The tetrameric M2 does not bind 3,3',5-triiodo-L-thyronine (T3). Binding of p58 to T3 and its analogs resulted in the inhibition of its pyruvate kinase activity. The apparent K; values of T3, L-thyroxine, and D-T3 are 30 aM, 100 nM, and 2 mM, respectively. L-Thyronine and 3,3',5'-triiodo-L-thyronine had no effect. This order of activity correlates with the thermogenic effects reported for T3 and its analogs. Conversion of p58 to the tetramer is reversible and is under the control of fructose 1,6-bisphosphate. The conversion is inhibited by T3 in a dose-dependent manner. Since pyruvate kinase is a key enzyme in regulating cellular ADP, ATP, and pyruvate, our findings suggest that p58 may be involved in mediating some of the cellular metabolic effects induced by thyroid hormones.The thyroid hormone 3,3',5-triiodo-L-thyronine (T3) plays an essential role in maintaining fetal and neonatal development, regulating amino acid and electrolyte transport into the cell, modulating carbohydrate, protein, and lipid metabolism, and increasing the rate of oxidative phosphorylation. No single mechanism has been found to account for all the diverse actions of-thyroid hormones.Some of the thyroid hormone effects are initiated through the interaction of T3 with nuclear receptors (see reviews in refs. 1 and 2). However, some ofthe effects of T3, such as the enhancement of enzymatic activity (3,4) and cellular amino acid accumulation and ATP synthesis (5) that occur in the absence of protein synthesis, have to be accounted for by alternative mechanism(s). Therefore, extranuclear T3 binding sites that might mediate such activities have been sought. Cheng and coworkers (6) purified a cytosolic thyroid hormone-binding protein (p58) to homogeneity. The purified protein retains its T3-binding activity and specificity (6), and two monoclonal antibodies against p58 have been developed (7). p58 has an apparent molecular weight of 60,000 by gel filtration and 58,000 by SDS/PAGE. To study its cellular function and the roles it plays in T3 action, we isolated and sequenced the cDNA that encodes p58A We found that p58 is a monomer of pyruvate kinase (ATP:pyruvate 02-phosphotransferase, EC 2.7.1.40) subtype M2 and that its conversion to the tetrameric pyruvate kinase is regulated by fructose 1,6-bisphosphate (Fru-1,6-P2).

A method-"fracture label"-is described for the cytochemical labeling of the membrane faces produced by freeze-fracture. Human erythrocytes embedded in a crosslinked matrix are frozen, fractured in liquid nitrogen, thawed, labeled, and cut into thin sections. Electron microscope observation of the fracture faces shows preferential partition of concanavalin A binding sites with the. inner half of the membrane. This signifies that, during freeze-fracture, binding sites are dragd from the outer surface across the outer ("exoplasmic") half of the membrane and retained on the protoplasmic fracture face (face P). The fracture process results in exposure of new. anionic-sites on face P. Fracture-label can be applied to the cytochemical characterization of the cellular components exposed. by freeze-fracture of isolated cells and tissues.Freeze-fracture splits membranes along their hydrophobic interior, following the juncture ofend groups provided by a membrane continuum with bilayer organization (1-4). Although fracture of the bilayer produces smooth faces, in all biological membranes these faces are interrupted by particulate components ("membrane intercalated particles" or "intramembranous particles") and, in most, by subtler rugosities ["subparticles" (5)]. Combined use ofimmunochemical and cytochemical techniques with freeze-etching methods, and freeze-fracture observation of reconstituted membrane preparations, demonstrate that, in the systems used, the particles represent the sites ofintegral transmembrane proteins (6-12). Because of the qualitative similarity offreeze-fracture images produced by all biological membranes, the particles are assumed to represent local structural asymmetries in the fracture process provoked by integral membrane.proteins and, possibly, their-associated lipids (3, 4). In the erythrocyte membrane the particles represent the site of the two main integral transmembrane proteins (glycophorin and band III component) which bear AB(H) antigens, influenza virus receptors, wheat germ agglutinin and concanavalin A (Con A) binding sites, as well as anionic sites (8)(9)(10)(11)(13)(14)(15).Although splitting of the bilayer continuum by freeze-fracture (and, in consequence, the partition of peripheral membrane proteins) is established, less is known about the fracture behavior of integral membrane proteins (3, 16)-in particular, their antigens and lectin binding sites at the surface (17). Most particles are generally associated with the inner membrane half (1, 2)-i. e., they are observed on the protoplasmic or P face (18). This may be the result ofa stochastic process or reflect individual differences of fracture behavior of the components in each particle or both. On face P the particles represent, at least in part, portions of integral proteins located at the outer (exoplasmic, E) membrane half. It is unknown whether the particles contain hydrophilic surface groups ofintegral proteins dragged, during fracture, from the outer surface. We have labeled freezefractured erythrocytes and repor...

Applications of the new fracture-labeling techniques for the observation of cytochemical labels on platinum-carbon replicas are described. Frozen cells, embedded in a cross-linked protein matrix, and frozen tissues are fractured with a scalpel under liquid nitrogen, thawed, labeled, dehydrated by the critical point drying method, and replicated. This method allows direct, high-resolution, two-dimensional chemical and immunological characterization of the cellular membranes in situ, as well as detection of sites within cross-fractured cytoplasm and extracellular matrix.

Experimental details of a new method for the cytochemical characterization of the membrane faces and cytoplasm produced by freeze-fracture of isolated cells and tissues are presented. This new method-"fracture-label"-involves grinding of frozen samples immersed in liquid nitrogen, thawing, cytochemical labeling of the fractured faces, and processing for thin section electron microscopy. Cationized ferritin (at pH. 7.5 and 4.0), colloidal iron, as well as concanavalin A are used to label the fracture faces of leukocytes and HeLa cells embedded in a cross-linked matrix of bovine serum albumin and of liver and spleen tissues. Our results show the presence of numerous anionic binding sites on the fracture faces of all plasma and cytoplasmic membranes, and of concanavalin A binding sites prefer-Literature Cited