Reticulocytes, like other cells, selectively degrade certain abnormal proteins by an energy-dependent yrocess. When isolated rabbit reticulocytes incorporate the vaSme analog 2-amino4--chlorobutyric acid (ClAbu) in place of valine, they produce an abnormal globin that is degraded with a halflife of 15 min. Normal hemoglobin, in contrast, undergoes little or no breakdown within these cells. Cell-free extracts from reticulocytes have been shown to rapidly hydrolyze these abnormal proteins. The degradative system is located in the 100,000 X g supernatant, has a pH optimum of 7.8, and does not appear to of 1ysosomal origin. This breakdown of analog-ontaining protein was stimulated severalfold by ATP, and slightly by ADP. AMP and adenosine-3':5'-cyclic monophosphate had no significant effect on proteolysis. Experiments with ATP analogs suggest that the terminal high energy phosphate is important in the degradative process.Proteolysis in the cell-free system and in intact reticulocytes was inhibited-by the same agents (L-1-tosylamido-2-phenylethylchloromethyl ketone, N *-tosyl-Llysine chloromethyl ketone, N-ethylmaleirnide, iodoacetamide, and o-phenanthroline) In addition, the relative rates of degradationof several polypeptides in the cell-fee extracts paralleled degradative rates within cells. Thus, a soluble nonlysosomal proteolytic system appears responsible for the energy-dependent degradation of abnormal proteins in reticulocytes.Proteins within animal and bacterial cells are continuously being degraded to amino acids, and their rates of degradation in part determine their intracellular concentrations (1-4). Proteins with abnormal structures are degraded especially rapidly (-3, 5-8) and this process prevents the accumulation of potentially harmful polypeptides (1, 2). Despite the physiological significance of protein catabolism, the responsible enzymes and degradative pathways are still unknown. In animal cells, most workers have assumed that the lysosome is the site of protein degradation, because of the high concentration of proteases within this organelle and because the lysosome is involved in the degradation of endocytosed proteins (9, 10). However, various nonlysosomal proteases also exist and may well be involved in the breakdown of cell proteins (2,11,12). One reason for the lack of knowledge of the degradative pathways is that cell-free preparations have generally failed to display important characteristics of intracellular proteolysis. For example, a puzzling feature of this process is that inhibitors of energy metabolism which severely reduce cellular ATP levels also inhibit protein degradation (2, 7). To explain this effect, previous workers have suggested an energy requirement for transport of substrate into the lysosome (2, 10) or for maintaining an acid milieu within this organelle (2, 13). However, an energy requirement for protein degradation also has been demonstrated in bacteria which lack lysosomes (2, 7). In addition, cell-free preparations have often failed to degrade proteins tha...

Chronic treatment of rats with clenbuterol, a beta 2-receptor agonist (8-12 wk), caused hypertrophy of histochemically identified fast- but not slow-twitch fibers within the soleus, while the mean areas of both fiber types were increased in the extensor digitorum longus (EDL). In contrast, treatment with the beta 2-receptor antagonist, butoxamine, reduced fast-twitch fiber size in both muscles. In the solei and to a lesser extent in the EDLs, the ratio of the number of fast- to slow-twitch fibers was increased by clenbuterol, while the opposite was observed with butoxamine. The muscle fiber hypertrophy observed in the EDL was accompanied by parallel increases in maximal tetanic tension and muscle cross-sectional area, while in the solei, progressive increases in rates of force development and relaxation toward values typical of fast-twitch muscles were also observed. Our results suggest a role of beta 2-receptors in regulating muscle fiber type composition as well as growth.

In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range 200 -400 kDa ("statosome I") and 1-2 MDa ("statosome II"). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from 200 -400 kDa to 1-2 MDa; (ii) a small pool of monomeric STAT3 and tyrosinephosphorylated STAT3 eluting at 80 -100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") coeluted with catalase at 230 kDa. In order to identify the protein components of the 200 -400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from 20 to 114 kDa co-shifted with STAT3; three of these (p60, p20a, and p20b) were co-shifted in an IL-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major IL-6-dependent STAT3-coshifted p60 protein as the chaperone GRP58/ER-60/ ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.

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