Protein phosphorylation in isolated mitochondria and the effects of protein kinase C.

When isolated intact rat liver mitochondria are incubated with [gamma-32P]ATP the major phosphorylated proteins are those of 47 and 36 kDa. Phosphorylation of the 47 kDa protein, but not of the 36 kDa protein, is inhibited by carboxyatractyloside, an inhibitor of mitochondrial ATP uptake, while phosphorylation of the 36 kDa protein is inhibited by various uncouplers and an inhibitor of mitochondrial respiration. Addition of purified protein kinase C to the isolated mitochondria leads to the phosphorylation of 69, 37 and 17 kDa proteins. As with other substrates for protein kinase C, phosphorylation of these proteins is dependent on Ca2+ and markedly stimulated by various tumor promoters.

Cellular uptake and localization of fluorescent derivatives of phorbol ester tumor promoters.

The synthetic fluorescent derivatives of 12-O-tetradecanoylphorbol-13-acetate (TPA), dansyl-TPA, dansyl-TPA-20-acetate and dansyl-TPA-13-desacetate, have ID50 values in the [3H]PDBu binding assay of 2nM, 30nM and 1000nM respectively; the ID50 value of TPA is 4nM. Dansyl-TPA is also equipotent with TPA as an activator of protein kinase C(PKC) producing half maximum stimulation at 2nM. Dansyl-TPA-13-desacetate is almost as potent as dansyl-TPA, while dansyl-TPA-20-acetate is completely inactive as an activator of PKC. The cellular uptake of these fluorescent TPA derivatives tends to parallel their activity in the [3H]PDBu binding assay. Treatment of C3H 10T1/2 cells with 100nM dansyl-TPA results in intense fluorescence of the entire cytoplasm, while the nucleus is virtually devoid of fluorescence. The uptake of fluorescence is quenched by an excess of TPA. Thus, dansyl-TPA rapidly enters cells and binds to specific sites distributed throughout the cytoplasm. Presumably these sites reflect the cellular localization of phorbol ester receptors and protein kinase C.

Activation of protein kinase C by tumor promoting phorbol esters, teleocidin and aplysiatoxin in the absence of added calcium.

We have found that maximum stimulation (greater than 10-fold) of kinase activity of a bovine brain preparation of calcium- and phospholipid-dependent protein kinase (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) occurs in the presence of phospholipid, but in the absence of added Ca2+. In effect, nM concentrations of TPA substitute for mM concentrations of added Ca2+, and the two agents are not synergistic. Biologically active analogs of TPA such as phorbol-12,13-dibutyrate (PDBu), 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA) or mezerein were also effective activators of PKC, as were the chemically unrelated tumor promoters teleocidin and aplysiatoxin, when tested at nM concentrations in the absence of added Ca2+. On the other hand, the biologically inactive compounds phorbol, 4-alpha-phorbol-12,13-didecanoate (4-alpha-PDD), HHPA-13,20-diacetate and 1,2-dihydro-20-deoxy-HHPA did not affect PKC activity in the absence or presence of Ca2+. Our results are consistent with a stereochemical model in which the hydrophilic domains of certain diterpenes, teleocidin and aplysiatoxin interact specifically with PKC apoenzyme, while their hydrophobic domains interact with phospholipid, thus forming an enzymatically active ternary complex.

Preparation and properties of nickel hemoglobin.

Hemoglobin A reconstituted with nickel protoporphyrin IX (NiHbA) has been prepared and characterized. Kinetics of its reaction with p-mercuribenzoate and with haptoglobin, absorption and circular dichroism spectra, and x-ray crystallographic properties have been investigated as probes of its structural conformation. The results suggest that NiHbA exists in a structure that is similar to the deoxy, or T-state of HbA. It is proposed that NiHbA and its derivatives may serve as a useful model for future studies of hemoglobin allosteric changes.

Hemoglobin-binding site on haptoglobin probed by selective proteolysis.

Selective proteolysis has been used to delineate the hemoglobin-binding site on haptoglobin heavy chain. Haptoglobin was cleaved specifically by plasmin, trypsin, chymotrypsin, staphylococcal protease, and thermolysin. Haptoglobin-hemoglobin complex was treated with these enzymes to determine which sites were protected from cleavage by the hemoglobin. The modified haptoglobins were tested for changes in their hemoglobin and hemoglobin alpha chain-binding properties. The sites of proteolytic cleavage were identified from the newly generated NH2 termini by automated Edman degradation amino acid-sequencing techniques. The results suggest that residues 128 through 131, 136 and 137, as well as 9 and 10 of the heavy chain may be involved in the binding of hemoglobin. On the other hand, residues 159 and 160, which lie in the 17-residue additional loop that is unique to haptoglobin among its homologous serine protease family, and residues 73 and 74, which lie close to the carbohydrate-binding residues, appear to be remote from the hemoglobin-binding site.

Hemoglobin binding site and its relationship to the serine protease-like active site of haptoglobin.

Haptoglobin forms a stable, irreversible complex with hemoglobin. The H chain of haptoglobin, which is the subunit that binds hemoglobin, shows strong sequence homology with the serine protease family. This raises the question of whether hemoglobin binds to the protease-like active site pocket of H chain as the protease inhibitors do with serine proteases. This question can be tested by binding proflavin and thionin to haptoglobin because these dyes are known to interact specifically with serine proteases at the peptide binding site. A single, specific binding site, characteristic of the serine proteases, was found for haptoglobin with association constants for proflavin or 1.4 x 10(3) at pH 7.1 and 8.2 x 10(3) at pH 9.5 and for thionin of 3.5 x 10(3) at pH 7.1. In order to confirm that these dyes are indeed binding to the specificity pocket of haptoglobin, competition experiments with classical serine protease substrates and inhibitors were performed. The results showed that trypsin-specific substrates and inhibitors did compete with proflavin binding, as expected from the homology, and that reagents of a chymotryptic specificity did not. When the dye titrations were performed on haptoglobin-hemoglobin complex, the same binding constants were obtained as for haptoglobin alone. This demonstrates that the active site-like region of haptoglobin and the hemoglobin binding site are mutually exclusive and do not interact in any way.