The shedding activity of ADAM17 is sequestered in lipid rafts

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.     

Evidence that bacteria induce translocation of protein kinase C activity in human polymorphonuclear leukocytes

Particulate fraction associated protein kinase activity was studied in human polymorphonuclear leukocytes stimulated by bacteria. Staphylococcus aureus was found to increase particulate fraction associated protein kinase C activity in a time and concentration dependent manner. The increase comprised both the phospholipid dependent and independent kinase activity and was augmented by addition of serum. Similar observations were done using Staphylococcus epidermidis and Klebsiellae pneumoniae. However, Escherichia coli only increased the phospholipid independent kinase activity in the particulate fraction, which suggests the presence of protease activity.     

Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.     

The effects of leucocyte elastase on the mechanical properties of adult human articular cartilage in tension

The effects of leucocyte elastase on the tensile properties of adult human articular cartilage were examined in detail in 99 specimens from hip, knee and ankle joints in the age range 16–83 years. The results showed that elastase reduced the tensile stiffness of cartilage, both at low stress and at fracture. The tensile strength of cartilage was also considerably reduced by the action of elastase. Biochemical analysis of the incubation media, and the specimens, revealed that 90%, or more, of the proteoglycan was released from the cartilage, whilst the release of collagen was negligible. Leucocyte elastase is known to degrade the non-helical terminal peptides of cartilage collagen molecules and thereby disrupt the main intermolecular cross-links in collage fribrils. A previous study (Kempson, G.E., Tuke, M.A., Dingle, J.T., Barrett, A.J. and Horsfield, P.H. (1976) Biochim. Biophys. Acta 428, 741–760) showed the lack of effect of proteoglycan degradation alone on the tensile strength and stiffness of cartilage. The reduction in strength and stiffness recorded in the present study can, therefore, be attributed to the action of elastase on the collagen in cartilage and it emphasises the important of covalent intermolecular cross-links to the mechanical properties of collagen fibrils.     

The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.     

Photoaffinity labeling of the N-formyl peptide receptor of human polymorphonuclear leukocytes

Quantitative analysis of ligand-occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor-ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 approximately 0.7 nM). When incubated at 0 degree C with whole PMNL, radioactive ligand became specifically and saturably associated with a 60-70,000-dalton species (as assessed by SDS-PAGE) after exposure to UV light. Addition of 10-100-fold excess of unlabeled parent or unlabeled azidopeptide derivative completely blocked uptake into this species. Approximately 20-40% of the available surface receptor-binding sites were covalently labeled under these conditions. Subcellular fractionation of the labeled cells on sucrose gradients after homogenization showed that the labeled species was primarily associated with plasma membrane-rich fractions. The labeled receptor could be completely solubilized with Triton X-100 in a form which eluted as a single species with a Stoke's radius of less than 50 A on Sepharose 4B columns. In addition, the solubilized receptor-ligand complex bound specifically to wheat germ agglutinin, indicating that it is probably a glycoprotein. The ability to label the receptor in living PMNL with a high efficiency should facilitate the study of receptor dynamics and receptor physiochemical properties in this system.     

Evidence that glutamine is involved in neutrophil function

Phosphate-dependent glutaminase (PDG) activity, a key enzyme of glutamine metabolism, was determined in neutrophils obtained from the intra-peritoneal cavity (PC) or bronchoalveolar space (BAS) after administration of 1 ml or 100 microl, respectively of saline, glycogen solution (1%) or lipopolysaccharide (LPS 0.1 mg (100 microl)(-1)). Neutrophils were obtained by lavage of both sites with 20 ml saline 24 h after the administration of the stimuli. Glycogen and LPS, depending on the site the cells were obtained from, differently modulated PDG activity. Cells from BAS stimulated by glycogen or LPS had raised PDG activity to 30.5 +/- 5.2 and 42.7 +/- 12.1 nmol min(-1) mg(-1) protein, respectively, when compared with saline (9.1 +/- 0.9 nmol min(-1) mg(-1) protein); mean +/- SEM. On the other hand, cells from PC showed different PDG activity: 52.0 +/- 12.6 nmol min(-1) mg(-1) for saline, 36.5 +/- 9.5 nmol min(-1) mg(-1) for glycogen, and 76.6 +/- 11.2 nmol min(-1) mg(-1) for LPS; mean +/- SEM. Therefore, PDG activity varies with the site from which neutrophils are obtained and the stimulus imposed. The effect of glutamine on nitric oxide (NO) and tumour necrosis factor (TNF) production by peritoneal neutrophils, obtained after glycogen administration, cultured in the presence of LPS (0.5 microg ml(-1)) was also examined. The addition of glutamine at concentrations varying from 2 to 20 mM did not markedly affect NO production. Glutamine alone at 2 mM did not modify the production of TNF but in the presence of LPS caused a significant decrease. So, glutamine may preserve the function of neutrophils during infections and injuries.     

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.     

Effect of microtubule-disrupting agents on superoxide production in human polymorphonuclear leukocytes

We explored the effects of compounds known or proposed to affect microtubule functions on superoxide (O₂⁻) production in human polymorphonuclear leukocytes stimulated by N-formyl-methionyl-phenylalanine (f-Met-Phe), calcium ionophore A23187 and phorbol myristate acetate. F-Met-Phe-induced O₂⁻ production was markedly potentiated not only by microtubule-disrupting agents, including colchicine, vincristine, vinblastine, nocodazole, podophyllotoxin and griseofulvin, but also deuterium oxide (²H₂O), which is proposed to stabilize microtubules, and not affected by lumicolchicine. Ionophore A23187-induced O₂⁻ production was not influenced by colchicine, and markedly enhanced by ²H₂O, whereas phorbol myristate acetate-induced O₂⁻ production was not influenced by colchicine, and slightly inhibited by ²H₂O. ²H₂O did not counteract the effects of colchicine and vice versa. Dibutyryl cyclic AMP and prostaglandin E₁ inhibited O₂⁻ production stimulated by f-Met-Phe and ionophore A23187, whereas phorbol myristate acetate-induced O₂⁻ production was strongly resistant to the inhibitory effect of these agents. The enhancing effect of colchicine and ²H₂O on f-Met-Phe-induced O₂⁻ production was abolished by dibutyryl cyclic AMP. Colchicine promoted concanavalin A cap formation, and ²H₂O produced cancanavalin A patch formation, whereas dibutyryl cyclic AMP did not affect the distribution of concanavalin A receptors. In addition, ²H₂O and dibutyryl cyclic AMP did not interfere with the colchicine-induced concanavalin A cap formation. These findings suggest that f-Met-Phe, ionophore A23187 and phorbol myristate acetate may activate the oxidative metabolism of human polymorphonuclear leukocytes through different mechanisms, and that microtubule-disrupting agents, ²H₂O and cyclic AMP agonists may affect the different steps of the activating system of NAD(P)H oxidase.     

Odontoclastic resorption of the superficial nonmineralized layer of predentine in the shedding of human deciduous teeth

Resorption by odontoclasts of a superficial nonmineralized layer of predentine that occurs in prior to the shedding of human deciduous teeth was studied by light and electron microscopy. As resorption of the tooth roots neared completion, multinucleate cells appeared on the predentine surface of the coronal dentine between the degenerated odontoblasts, excavated characteristic resorption lacunae in the nonmineralized predentine. These multinucleate cells had the same ultrastructural characteristics as odontoclasts and histochemical demonstration of tartrate-resistant acid phosphatase activity in the multinucleate cells revealed intense staining in numerous small granules identified as lysosomes. Occasionally, the multinucleate cells simultaneously resorbed both nonmineralized and calcospherite-mineralized matrix in the predentine. The study demonstrates that multinucleate odontoclasts can resorb nonmineralized predentine matrix in vivo, probably in the same way as they resorb demineralized organic matrix in the resorption zone underlying their ruffled border.     

Evidence that NADPH is the actual substrate of the oxidase responsible for the "respiratory burst" of phagocytosing polymorphonuclear leukocytes

The relationship between glucose metabolism and the "respiratory burst" of phagocytosing polymorphonuclear leukocytes (PMN) was studied in a Renex 30-treated cell system of guinea pig PMN by a polarometric technique. Phagocytosing PMN were treated with a detergent (Renex 30) and recovery of respiratory activity was examined by addition of various concentrations of NADP and glucose-6-phosphate (G6P) to determine the availability of endogenously formed NADPH via the hexose monophosphate (HMP) pathway. The oxygen uptake by phagocytosing PMN ceased after the treatment with Renex 30 and was restored by the addition of NADP and G6P. Furthermore, the restoration of oxygen uptake was linearly proportional to the rate of NADPH formation on increase in either NADP or G6P concentration. Resting PMN showed no respiratory activity even in the presence of excess NADP and G6P, in which NADPH was formed at the same rate as in phagocytosing PMN. In a parallel experiment, recovery of respiratory activity was examined in the same system by addition of NAD and glyceraldehyde-3-phosphate (G3P) in that order to clarify whether the respiratory enzyme can utilize NADH formed via the glycolytic pathway. In contrast to the results in the NADPH-forming system, the addition of NAD and G3P induced slight oxygen uptake of Renex 30-treated PMN, but there was no difference in the oxygen uptake between resting and phagocytosis-activated PMN. The results indicated that the primary oxidase responsible for the "respiratory burst" is NADPH oxidase, and that its activity is coupled with glucose oxidation via the HMP pathway without the participation of other metabolic pathways such as glycolysis.     

The role of agonists that activate adenylate cyclase in the control of cAMP metabolism and enzyme release by human polymorphonuclear leukocytes

An inverse relationship between cAMP content and effector function is ascribed generally to immune and inflammatory cells. Previous reports imply, however, that human polymorphonuclear leukocytes (PMN) are less responsive than other inflammatory cells to adenylate cyclase (AC) agonists. We therefore examined the effects of isoproterenol, prostaglandin E1 (PGE1), adenosine, and histamine on the adenosine 3',5'-monophosphate (cAMP) content of PMN and on particle-stimulated lysosomal enzyme release. For comparison, the effect of AC agonists on the cAMP content of human peripheral lymphocytes was evaluated in parallel. Although potent stimuli for cAMP accumulation in lymphocytes, the AC agonists produced only marginal increases in the cAMP content of PMN; this difference in responsiveness was independent of agonist concentration or length of incubation. Inhibition of lysosomal enzyme release by the AC agonists was likewise marginal (< 20%). The addition of theophylline with isoproterenol produced additive inhibition without significant cAMP increases. Hydrocortisone, which caused a small increase in the cAMP content, markedly potentiated the effects of AC agonists on the cAMP level in PMN; the synergistic increases in cAMP were accompanied by additive effects on lysosomal enzyme release. It is concluded that human lymphocytes and PMN exhibit differential sensitivity to AC agonists and that this difference may provide a basis for the selective modulation of individual PMN- or lymphocyte-mediated events.     

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.     

Differences in the substrate binding sites of murine and human proteinase 3 and neutrophil elastase

Understanding the differences between murine (m) and human (h) proteinase 3 (PR3) and neutrophil elastase (NE) is crucial for the interpretation of in vivo studies of inflammatory processes. We built structural models of mPR3 and mNE and analyzed their surface properties. We performed molecular dynamics (MD) simulations on several enzyme-peptide complexes to investigate their interaction patterns. The analysis of trajectories confirms that murine and human complexes have different interaction patterns with peptidic substrates. We provide a map of the binding sites of the murine proteases and suggest sequence motifs that we predict to be specific for mPR3 or mNE.     

Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols

Binding of chemoattractants to receptors on human polymorphonuclear leukocytes (PMN) stimulates the phosphodiesteric cleavage of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and 1,2-diacylglycerols. To investigate the possible second messenger function of diacylglycerols in PMN activation, we tested the ability of a series of synthetic sn 1,2-diacylglycerols, known to stimulate protein kinase C in other systems, to promote superoxide anion release, oxygen consumption, lysosomal enzyme secretion, and chemotaxis. None of the diacylglycerols initiated the chemotactic migration of PMN. Several of the diacylglycerols however, were, active in stimulating superoxide anion release and lysozyme secretion, with dioctanoylglycerol (diC8) being the most potent. Unexpectedly, didecanoylglycerol (diC10) induced lysosomal enzyme secretion, but failed to stimulate superoxide production or oxygen consumption. All other biologically active diacylglycerols tested displayed similar EC50 for stimulating lysozyme secretion and superoxide production. The ability of the diacylglycerols to compete for phorbol dibutyrate (PDBu) binding in intact PMN suggested a mechanism for the divergent biological activity of diC10. Although the compounds that stimulated both superoxide production and lysosomal enzyme secretion competed for essentially all [3H]PDBu binding from its receptor, diC10, which only stimulated secretion, competed for 45% of the bound [3H]PDBu. Thus diacylglycerols can selectively activate certain functions of leukocyte chemoattractant receptor. The data suggest that a discrete pool of protein kinase C may mediate activation of the respiratory burst in PMN.     

Omega-oxidation is the major pathway for the catabolism of leukotriene B4 in human polymorphonuclear leukocytes

Leukotriene B4 (LTB4), formed by the 5-lipoxygenase pathway in human polymorphonuclear leukocytes (PMN), may be an important mediator of inflammation. Recent studies suggest that human leukocytes can convert LTB4 to products that are less biologically active. To examine the catabolism of LTB4, we developed (using high performance liquid chromatography) a sensitive, reproducible assay for this mediator and its omega-oxidation products (20-OH- and 20-COOH-LTB4). With this assay, we have found that human PMN (but not human monocytes, lymphocytes, or platelets) convert exogenous LTB4 almost exclusively to 20-OH- and 20-COOH-LTB4 (identified by gas chromatography-mass spectrometry). Catabolism of exogenous LTB4 by omega-oxidation is rapid (t1/2 approximately 4 min at 37 degrees C in reaction mixtures containing 1.0 microM LTB4 and 20 X 10(6) PMN/ml), temperature-dependent (negligible at 0 degrees C), and varies with cell number as well as with initial substrate concentration. The pathway for omega-oxidation in PMN is specific for LTB4 and 5(S),12(S)-dihydroxy-6,8,10,14-eicosatetraenoic acid (only small amounts of other dihydroxylated-derivatives of arachidonic acid are converted to omega-oxidation products). Even PMN that are stimulated by phorbol myristate acetate to produce large amounts of superoxide anion radicals catabolize exogenous leukotriene B4 primarily by omega-oxidation. Finally, LTB4 that is generated when PMN are stimulated with the calcium ionophore, A23187, is rapidly catabolized by omega-oxidation. Thus, human PMN not only generate and respond to LTB4, but also rapidly and specifically catabolize this mediator by omega-oxidation.     

Identification of an aldehyde dehydrogenase in the microsomes of human polymorphonuclear leukocytes that metabolizes 20-aldehyde leukotriene B4

We have previously reported that cytochrome P-450LTB in the microsomes of human polymorphonuclear leukocytes (PMN) catalyzes three omega-oxidations of leukotriene B4 (LTB4), leading to the sequential formation of 20-OH-LTB4, 20-CHO-LTB4, and 20-COOH-LTB4 (Soberman, R.J., Sutyak, J.P., Okita, R.T., Wendelborn, D.F., Roberts, L.J., II, and Austen, K. F. (1988) J. Biol. Chem. 263, 7996-8002). The identification of the novel final intermediate, 20-CHO-LTB4, allowed direct analysis of its metabolism by PMN microsomes in the presence of adenine nucleotide cofactors. Microsomes in the presence of 100 microM NAD+ or 100 microM NADP+ converted 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 with a Km of 2.4 +/- 0.8 microM (mean +/- S.E., n = 4) and a Vmax of 813.9 +/- 136.6 pmol.min-1.mg-1, for NAD+, as compared to 0.12 microM and 5.0 pmol.min-1.mg-1 (n = 2) for NADPH as a cofactor. The conversion of 1.0 microM of 20-CHO-LTB4 to 20-COOH-LTB4 in the presence of saturating concentrations (1.0 mM) of both NAD+ and NADP+ was not greater than the reaction in the presence of 1.0 mM of each cofactor separately, indicating that NAD+ and NADP+ were cofactors for the same enzyme. Antibody to cytochrome P-450 reductase did not inhibit the conversion of 20-CHO-LTB4 to 20-COOH-LTB4. When 1.0 microM 20-OH-LTB4 was added to microsomes in the presence of NADPH, approximately three-fourths of the product formed (63.7 +/- 5.1 pmol; mean +/- S.E., n = 3) was 20-CHO-LTB4 and approximately one-fourth (21.3 +/- 3.9 pmol; mean +/- S.E., n = 3) was 20-COOH-LTB4. In the presence of both NADPH and NAD+, only 20-COOH-LTB4 (85.5 +/- 9.9 pmol; mean +/- S.E., n = 3) was formed. PMN microsomes also contain an NADH-dependent aldehyde reductase which converts 20-CHO-LTB4 to 20-OH-LTB4, a member of the LTB4 family of molecules with biological activity. Based upon kinetic, cofactor and inhibition data, microsomal aldehyde dehydrogenase preferentially regulates the final and irreversible inactivation step in the LTB4 metabolic sequence.     

Evidence that high mobility group protein 17 is not phosphorylated in human colon carcinoma cells

The high mobility group proteins 14 and 17 were reported previously to be phosphorylated in murine and human tumor cell lines. Recently, it was suggested that subgroups of HMG-14, HMG-14a and 14b, but not HMG-17, were phosphorylated in situ in HeLa cells. In order to definitively determine whether HMG-17 is indeed phosphorylated or whether the protein previously identified as [³²P]HMG-17 was a subgroup of HMG-14, we have used the technique of electroblotting in conjunction with an immunochemical procedure utilizing anti-HMG-17 IgG. Our results indicate that HMG-17 was not phosphorylated in human colon carcinoma cell line HT-29 incubated for 18 h with ³²P_i, but that HMG-14a and HMG-14b were phosphorylated. In contrast, HMG-14a, -14b and -17 were phosphorylated in vitro in isolated nuclei incubated with [γ-³²P]ATP.