COOH-terminal Site Research Articles

The Role of Heat Shock Protein -90 (HSP-90) in Inflammatory Disorders

In human cells, Heat Shock Protein-90 (HSP-90) is present in the cytosol, nucleoplasm, endoplasmic reticulum, and mitochondria. The eukaryotic HSP-90 is multifunctionary and essential for cell viability, signal transduction, cell-cycle control as well as transcriptional regulation. The intracellular environment does not restrict HSP-90. It has a vital role in all types of inflammatory disorders, including cancer, autoimmune diseases, infectious inflammatory conditions. Hence, pharmacological inhibition of HSP-90 is currently a choice of therapeutic target for the treatment of autoimmune diseases, cancer, and infectious diseases. Based on the biology of HSP-90, several COOH-terminal ATPase sites of HSP-90, NH2-terminal ATPase sites of HSP-90, and Histone deacetylase inhibitors are evaluated and classified under various groups. For the treatment of different inflammatory disorders, HSP-90 identified as a promising therapeutic target. The present review may guide researchers for evaluating the HSP-90 targeted pathway as a useful therapeutic target for inflammatory diseases, including cancers.

Regulation of Eag by Ca2+/calmodulin controls presynaptic excitability in Drosophila.

Drosophila ether-à-go-go ( eag) is the founding member of a large family of voltage-gated K+ channels, the KCNH family, which includes Kv10, 11, and 12. Concurrent binding of calcium/calmodulin (Ca2+/CaM) to NH2- and COOH-terminal sites inhibits mammalian EAG1 channels at submicromolar Ca2+ concentrations, likely by causing pore constriction. Although the Drosophila EAG channel was believed to be Ca2+-insensitive (Schönherr R, Löber K, Heinemann SH. EMBO J 19: 3263-3271, 2000.), both the NH2- and COOH-terminal sites are conserved. In this study we show that Drosophila EAG is inhibited by high Ca2+ concentrations that are only present at plasma membrane Ca2+ channel microdomains. To test the role of this regulation in vivo, we engineered mutations that block CaM-binding to the major COOH-terminal site of the endogenous eag locus, disrupting Ca2+-dependent inhibition. eag CaMBD mutants have reduced evoked release from larval motor neuron presynaptic terminals and show decreased Ca2+ influx in stimulated adult projection neuron presynaptic terminals, consistent with an increase in K+ conductance. These results are predicted by a conductance-based multicompartment model of the presynaptic terminal in which some fraction of EAG is localized to the Ca2+ channel microdomains that control neurotransmitter release. The reduction of release in the larval neuromuscular junction drives a compensatory increase in motor neuron somatic excitability. This misregulation of synaptic and somatic excitability has consequences for systems-level processes and leads to defects in associative memory formation in adults. NEW & NOTEWORTHY Regulation of excitability is critical to tuning the nervous system for complex behaviors. We demonstrate in this article that the EAG family of voltage-gated K+ channels exhibit conserved gating by Ca2+/CaM. Disruption of this inhibition in Drosophila results in decreased evoked neurotransmitter release due to truncated Ca2+ influx in presynaptic terminals. In adults, disrupted Ca2+ dynamics cripples memory formation. These data demonstrate that the biophysical details of channels have important implications for cell function and behavior.

Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells.

Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin [arginine vasopressin (AVP)], a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells, but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCDc14 cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 COOH-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK.

Complement Component 5a Receptor Oligomerization and Homologous Receptor Down-regulation

Most G-protein-coupled receptors (GPCRs) form di(oligo)-meric structures that constitute signaling and trafficking units and might be essential for receptor functions. Cell responses to complement C5a receptor (C5aR) are tightly controlled by receptor desensitization and internalization. To examine the implication of dimerization in C5aR regulation, we generated an NH(2)-terminally modified C5aR mutant, unable to bind C5a, and a phosphorylation-deficient mutant. Neither an intact NH(2) terminus nor the presence of COOH-terminal phosphorylation sites appeared to be required for the formation of C5aR dimers. Upon C5a stimulation, mutant receptors did not internalize when individually expressed. C5a stimulation of cells that co-expressed wild type C5aR together with either unliganded or phosphorylation-deficient mutant resulted in co-internalization of mutant receptors with C5aR. Unliganded GPCRs can be cross-phosphorylated within a heterologous receptor dimer or by second messenger-activated kinases. C5a stimulation of (32)P-labeled cells that co-expressed the unliganded mutant with either C5aR or the phosphorylation-deficient mutant did not induce phosphorylation of the unliganded mutant. We can thus postulate that, in the case of C5aR, the stimulation and phosphorylation of one monomer is enough to lead to dimer internalization. The existence and functional implication of di(oligo)mer formation may be important for an accurate C5aR down-regulation in pathological conditions.

Impaired Insulin Activation and Dephosphorylation of Glycogen Synthase in Skeletal Muscle of Women with Polycystic Ovary Syndrome Is Reversed by Pioglitazone Treatment

Insulin resistance is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). The molecular mechanisms underlying reduced insulin-mediated glycogen synthesis in skeletal muscle of patients with PCOS have not been established. We investigated protein content, activity, and phosphorylation of glycogen synthase (GS) and its major upstream inhibitor, GS kinase (GSK)-3 in skeletal muscle biopsies from 24 PCOS patients (before treatment) and 14 matched control subjects and 10 PCOS patients after 16 wk treatment with pioglitazone. All were metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry. Reduced insulin-mediated glucose disposal (P < 0.05) was associated with a lower insulin-stimulated GS activity in PCOS patients (P < 0.05), compared with controls. This was, in part, explained by absent insulin-mediated dephosphorylation of GS at the NH2-terminal sites 2+2a, whereas dephosphorylation at the COOH-terminal sites 3a+3b was intact in PCOS subjects (P < 0.05). Consistently, multiple linear regression analysis showed that insulin activation of GS was dependent on dephosphorylation of sites 3a+3b in women with PCOS. No significant abnormalities in GSK-3alpha or -3beta were found in PCOS subjects. Pioglitazone treatment improved insulin-stimulated glucose metabolism and GS activity in PCOS (all P < 0.05) and restored the ability of insulin to dephosphorylate GS at sites 2 and 2a. Impaired insulin activation of GS including absent dephosphorylation at sites 2+2a contributes to insulin resistance in skeletal muscle in PCOS. The ability of pioglitazone to enhance insulin sensitivity, in part, involves improved insulin action on GS activity and dephosphorylation at NH2-terminal sites.

The Phosphatase PHLPP Controls the Cellular Levels of Protein Kinase C

The life cycle of protein kinase C (PKC) is controlled by multiple phosphorylation and dephosphorylation steps. The maturation of PKC requires three ordered phosphorylations, one at the activation loop and two at COOH-terminal sites, the turn motif and the hydrophobic motif, to yield a stable and signaling-competent enzyme. Dephosphorylation of the enzyme leads to protein degradation. We have recently discovered a novel family of protein phosphatases named PH domain leucine-rich repeat protein phosphatase (PHLPP) whose members terminate Akt signaling by dephosphorylating the hydrophobic motif on Akt. Here we show that the two PHLPP isoforms, PHLPP1 and PHLPP2, also dephosphorylate the hydrophobic motif on PKC betaII, an event that shunts PKC to the detergent-insoluble fraction, effectively terminating its life cycle. Deletion mutagenesis reveals that the PH domain is necessary for the effective dephosphorylation of PKC betaII by PHLPP in cells, whereas the PDZ-binding motif, required for Akt regulation, is dispensable. The phorbol ester-mediated dephosphorylation of the hydrophobic site, but not the turn motif or activation loop, is insensitive to okadaic acid, consistent with PHLPP, a PP2C family member, controlling the hydrophobic site. In addition, knockdown of PHLPP expression reduces the rate of phorbol ester-triggered dephosphorylation of the hydrophobic motif, but not turn motif, of PKC alpha. Last, we show that depletion of PHLPP in colon cancer and normal breast epithelial cells results in an increase in conventional and novel PKC levels. These data reveal that PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation.

Aberrant Processing of Human Proislet Amyloid Polypeptide Results in Increased Amyloid Formation

The amyloid present in the islets of Langerhans in type 2 diabetes is polymerized islet amyloid polypeptide (IAPP). The precursor protein proIAPP is posttranslationally modified, a process involving the removal of NH2- and COOH-terminal flanking peptides. This step is performed by the prohormone convertases PC2 and PC1/3. PC2 processes proIAPP preferably at the NH2-terminal processing site, and PC1/3 processes proIAPP exclusively at the COOH-terminal site. Little is known regarding the exact circumstances leading to islet amyloid formation. In this study, we have examined the possible significance of aberrant processing of proIAPP on amyloid formation in several in vitro cellular systems. In our studies, human (h)-proIAPP was transfected into beta-TC-6 cells expressing both prohormone convertases and in which proIAPP is processed into IAPP. Additionally, h-proIAPP was transfected into three different pituitary-derived cell lines with different prohormone convertase profiles: AtT-20 cells (deficient in PC2), GH3 cells (deficient in PC1/3), and GH4C1 cells (deficient in both convertases). We followed the processing of h-proIAPP with antibodies specific for the respective cleavage sites and stained the cells with Congo red to verify the accumulation of amyloid. Incomplete processing of h-proIAPP that occurs in AtT-20 and GH4C1 cells resulted in the formation of intracellular amyloid. No amyloid developed in beta-TC-6 and GH3 cells lines with full processing of proIAPP. An intracellular increase in proIAPP and/or its metabolic products may thus promote intracellular amyloid formation, thereby causing cell death. When extracellularly exposed, this amyloid might act as template for continuing amyloid formation from processed IAPP released from the surrounding beta-cells.

Characterization of the Two eIF4A-binding Sites on Human eIF4G-1

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 x 10(5) m(-1) s(-1), 2.1 x 10(-3) s(-1), and 17 nm for the central site and 5.1 x 10(3) m(-1) s(-1), 1.7 x 10(-3) s(-1), and 330 nm for the COOH-terminal site.

Glycogen synthase sensitivity to insulin and glucose-6-phosphate is mediated by both NH2- and COOH-terminal phosphorylation sites.

In skeletal muscle, insulin activates glycogen synthase by reducing phosphorylation at both NH2- and COOH-terminal sites of the enzyme and by elevating the levels of glucose-6-phosphate, an allosteric activator of glycogen synthase. To study the mechanism of regulation of glycogen synthase by insulin and glucose-6-phosphate, we generated stable Rat-1 fibroblast clones expressing rabbit muscle glycogen synthase with Ser-->Ala substitutions at key phosphorylation sites. We found that 1) elimination of the phosphorylation of either NH2- or COOH-terminal sites did not abolish insulin stimulation of glycogen synthase; 2) mutations at both Ser-7 and Ser-640 were necessary to bypass insulin activation; 3) mutation at Ser-7, coupled with the disruption of the motif for recognition by glycogen synthase kinase-3 (GSK-3), did not eliminate the insulin effect; and 4) mutation of either Ser-7 or Ser-640 increased the sensitivity of glycogen synthase to glucose 6-phosphate >10-fold. We conclude that Ser-7 and Ser-640 are both involved in mediating the response of glycogen synthase to insulin and activation by glucose 6-phosphate. In Rat-1 fibroblasts, GSK-3 action is not essential for glycogen synthase activation by insulin, and GSK-3-independent mechanisms also operate.

A monoclonal antibody against the COOH-terminal site of α 2-antiplasmin reveals heterogeneity in the non-plasminogen binding form of α 2-antiplasmin

We have studied the plasminogen-binding and non-binding molecular forms of α2-antiplasmin (α2-AP), the major inhibitor of plasmin, using a modification of the crossed immunoelectrophoresis technique (mCIE). The previous technique using addition of Lys-plasminogen to the gel of the first dimension electrophoresis was simplified using commercially available monoclonal antibodies (MAb) against the COOH-terminal part of α2-AP, which harbours the plasminogen binding site. Indeed, the addition of MAb resulted in a mobility reduction of the plasminogen binding (PB) form of α2-AP, with an apparent Kd of 12.5 nM, whereas the non-plasminogen binding form (NPB-α2-AP) in plasma retained its normal mobility. The method with MAb was applied to study the molecular forms of α2-AP in plasma and serum of healthy individuals and of patients with various diseases as well as in other body fluids, such as ascites or synovial fluid. Upon investigation the conversion of PB-α2-AP into NPB-α2-AP in vitro, however we found evidence for a difference in specificity between the method using Lys-plasminogen and MAb. Though in-vitro-formed NPB-α2-AP loses its plasminogen binding, its MAb binding remains intact. This is in contrast to in vivo circulating NPB-α2-AP which has lost both properties. In selected samples of the conditions mentioned above we found no evidence for significant occurrence of the MAb binding NPB-α2-AP in vivo. We suggest therefore that the conversion in vivo and in vitro have different mechanisms and that the MAb is a useful tool for study of patient samples and for the further investigations into the mechanism of conversion.

Ca2+ Binding to Calmodulin and Its Role in Schizosaccharomyces pombe as Revealed by Mutagenesis and NMR Spectroscopy

As a first step toward identifying the important structural elements of calmodulin from Schizosaccharomyces pombe, we examined the ability of heterologous calmodulins and Ca(2+)-binding site mutant S. pombe calmodulins to replace the essential cam1+ gene. A cDNA encoding vertebrate calmodulin allows growth of S. pombe. However, calmodulin from Saccharomyces cerevisiae does not support growth even though the protein is produced at high levels. With one exception, all mutant S. pombe calmodulins with one or more intact Ca(2+)-binding sites allow growth at 21 degrees C. A mutant containing only an intact Ca(2+)-binding site 3 fails to support growth, as does S. pombe calmodulin with all four Ca(2+)-binding sites mutated. Several of the mutant proteins confer a temperature-sensitive phenotype. Analysis of the degree of temperature sensitivity allows the Ca(2+)-binding sites to be ranked by their ability to support fission yeast proliferation. Site 2 is more important than site 1, which is more important than site 4, which is more important than site 3. A visual colony color screen based on the fission yeast ade1+ gene was developed to perform these genetic analyses. To compare the Ca(2+)-binding properties of individual sites to their functional importance for viability, Ca2+ binding to calmodulin from S. pombe was studied by 1H NMR spectroscopy. NMR analysis indicates a Ca(2+)-binding profile that differs from those previously determined for vertebrate and S. cerevisiae calmodulins. Ca(2+)-binding site 3 has the highest relative affinity for Ca2+, while the affinities of sites 1, 2, and 4 are indistinguishable. A combination of an in vivo functional assay and an in vitro physical assay reveals that the relative affinity of a site for Ca2+ does not predict its functional importance.

Interactions between cAMP-dependent and SNF1 protein kinases in the control of glycogen accumulation in Saccharomyces cerevisiae.

The synthesis of glycogen in Saccharomyces cerevisiae is stimulated by nutrient limitation and requires both glycogen synthase and the glycogen branching enzyme. Of the two glycogen synthase genes present in yeast, GSY2 appears to be more important for the accumulation of glycogen upon entry into stationary phase. In cells grown on glucose, GSY2 mRNA levels increased approximately 10-fold during the transition from logarithmic to stationary phase. Growth of cells in glycerol, however, resulted in constitutive expression of GSY2 mRNA and the corresponding protein, GS-2, suggestive of glucose repression of GSY2. Mutants defective in the SNF1 gene, which encodes a protein kinase important in glucose repression mechanisms, are known not to accumulate glycogen. A modest 2-4-fold decrease in total GS-2 level was observed, and upon entry into stationary phase, the enzyme was blocked in the inactive, phosphorylated state in snf1 strains. The GS-2 protein is thought to be regulated by covalent phosphorylation of three COOH-terminal sites (Hardy, T.A., and Roach, P.J. (1993) J. Biol. Chem. 268, 23799-23805), removal of which results in constitutively active glycogen synthase that bypasses phosphorylation controls. Expression of COOH-terminally truncated GS-2 in snf1 cells restored glycogen accumulation, and so we propose that the SNF1 kinase controls the phosphorylation state of GS-2. Cyclic AMP pathways also exert control over glycogen accumulation. In bcy1 cells, which have constitutively active cyclic AMP-dependent protein kinase, greatly reduced levels of both GS-2 message and protein were observed. With wild type GSY2 placed under control of the ADH1 promoter, bcy1 cells did not accumulate glycogen despite increased GS-2. Overexpression of truncated GS-2, however, resulted in definite though reduced glycogen accumulation; the glycogen synthesized was structurally distinct from wild type with properties characteristic of less branched polysaccharide. We conclude that the cAMP pathway controls both the expression and the phosphorylation state of GS-2. Furthermore, other factor(s) necessary for glycogen biosynthesis, such as the branching enzyme GLC3, must also be under negative control by the cAMP pathway. The results demonstrate interactive controls of GS-2 by the cAMP-dependent and SNF1 protein kinases.

Rabbit skeletal muscle glycogen synthase expressed in COS cells. Identification of regulatory phosphorylation sites.

Rabbit skeletal muscle glycogen synthase contains multiple sites for phosphorylation. To investigate the relative importance of these sites, the enzyme was overexpressed in COS M9 cells, and Ser-->Ala mutations were introduced singly, or in combinations, at nine known phosphorylation sites. Overexpressed wild-type enzyme had a very low +/- glucose-6-P activity ratio of approximately 0.01, indicative that the glycogen synthase is in a highly phosphorylated state. No single Ser-->Ala mutation was able to cause a substantial increase in activity ratio; rather, simultaneous mutation at both NH2- and COOH-terminal sites was needed. The most effective combinations were mutations at site 3a (Ser-640) or site 3b (Ser-644) together with site 2 (Ser-7). The results were consistent with site 2 phosphorylation being a prerequisite for phosphorylation of site 2a (Ser-10). Mutation of site 5 (Ser-656) perturbed COOH-terminal phosphorylation but did not prevent inactivation. Expression of the most active mutants correlated with increased glycogen accumulation in the COS M9 cells. In summary, we conclude that (i) the sites most important for activating the enzyme are sites 2, 2a, 3a, and 3b; (ii) removal of phosphate from both NH2- and COOH-terminal sites is required for activation; and (iii) sites 3a and/or 3b can be phosphorylated in COS cells by mechanisms that do not depend on phosphorylation of site 5.

Ras signaling in the activation of glucose transport by insulin.

An approach involving microinjection and microanalysis has been developed to investigate signal-transduction pathways involved in the hormonal control of metabolism. We have applied this strategy to investigate the role of Ras signaling in the acute activation of glucose transport by insulin in cardiac myocytes. Glucose transport activity was assessed by measuring the initial rate of accumulation of 2-deoxyglucose 6-phosphate (dGlc6P) in individual cells after incubation in 2-deoxyglucose. Insulin increased accumulation of dGlc6P by 3- to 4-fold, consistent with its stimulatory effect on glucose transport. Accumulation of dGlc6P was increased severalfold by microinjecting the nonhydrolyzable GTP analogue, guanosine 5'-[gamma-thio]triphosphate, which activates members of the Ras superfamily of GTP-binding proteins. Injecting activated Ha-Ras protein also mimicked insulin by increasing dGlc6P; whereas, injecting a Ras protein lacking the COOH-terminal site of fatty acylation required for Ras function was without effect. Introducing the neutralizing Ras antibody Y13-259 into cells attenuated the effect of insulin. These findings implicate Ras in the acute regulation of metabolism by insulin.

Specific phosphorylation of a COOH-terminal site on the full-length form of the alpha 1 subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase.

The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.

Comparison of the antigenic peptides between human prostatic and lysosomal acid phosphatases

The relative antigenicity (capacity to bind antibodies raised against the intact prostatic acid phosphatase) of the selected peptides from human prostatic and lysosomal acid phosphatases was evaluated in a competitive assay. Both prostatic and lysosomal acid phosphatases were shown to possess similar antigenic determinants on both terminal regions, along with more similarity on NH2-terminal peptide than COOH-terminal site. At least one additional antigenic site is present at the internal region of prostatic acid phosphatase, since the mixture of both amino- and carboxyl-terminal peptides exhibited only 70% inhibition.

Sites of stromelysin cleavage in collagen types II, IX, X, and XI of cartilage.

Human recombinant stromelysin-1 was shown to cleave four types of collagen (types II, IX, X, and XI) prepared from bovine and rat cartilages at specific sites. Stromelysin-1 cleaved salt-soluble native molecules of type IX collagen into two main triple-helical fragments, COL1 and COL2,3. Protein microsequencing identified the exact cleavage sites in the NC2 domain of all three chains, alpha 1(IX), alpha 2(IX), and alpha 3(IX). Stromelysin-1 also acted as a "telopeptidase," in that it efficiently clipped intact molecules of types II and XI collagens at sites just inside their terminal cross-linking hydroxylysine residues. Native molecules of type X collagen were cleaved by stromelysin-1 within their triple helical domains at a COOH-terminal site that reduced the alpha 1(X) chain size by 10 kDa. These findings suggest an important role for stromelysin in the turnover and remodeling of the collagenous matrix of cartilage both normally and in degenerative joint disease.

The orientation of halorhodopsin in the cell membrane of halobacteria.

The orientation of the light-driven chloride pump, halorhodopsin, in the membrane was determined using antibodies directed against a synthetic peptide which represents the C-terminal segment of the protein. Antibodies against this decapeptide did not bind to right-side-out cell vesicles. Partial inversion by sonication or lysis under low salt conditions exposed this COOH-terminal antigenic site. Antibody binding was removed by preincubation with the decapeptide. The COOH terminus of the molecule is therefore located on the cytoplasmic surface of the membrane.

The influence of uncoordinated histidines on iron release from transferrin. A chemical modification study.

Histidine residues that influence the chelate-mediated removal of iron from transferrin have been investigated. Diferric human serum transferrin was chemically modified to various extents using ethoxyformic anhydride, a reagent for histidines. A kinetic analysis of the modification reaction revealed the presence of a fast reacting pool of 9 +/- .8 histidine residues and a slow reacting pool of 5.8 +/- .6 residues. There are 18 histidine residues in transferrin. The rates of modification of the two pools differed by a factor of 5. The pyrophosphate-mediated removal of iron from the two binding sites of native and partially modified transferrins was studied at pH 6.9 using desferrioximine B as a terminal iron acceptor. Under these conditions, the rate of iron removal from the NH2-terminal site was about six times faster than from the COOH-terminal site. Both rates were significantly reduced, i.e. by a factor of approximately 6-8, upon complete ethoxyformylation of all reactive histidines on the protein. The kinetic data of partially modified transferrins were analyzed by the Tsou Chen-Lu statistical method; the results are consistent with the hypothesis that modification of a single uncoordinated histidine in each of the two iron binding domains stabilizes the protein kinetically against loss of iron. The dependence of the iron removal reaction on pH is consistent with such an interpretation. The putative histidines, although not ligands, may be close to the metal in both binding sites, thus influencing the rate of iron removal by pyrophosphate. These histidines belong to the pool of rapidly modified residues and thus are readily accessible to solvent and chelators.

Localization of two epitopes of protein L7/L12 to both the body and stalk of the large ribosomal subunit. Immune electron microscopy using monoclonal antibodies.

Four molecules of ribosomal protein L7/L12 are found as two dimers on the Escherichia coli 50 S ribosomal subunit. Immune electron microscopy using monoclonal antibodies directed against two epitopes of protein L7/L12 has allowed placement of elements of each dimer. One monoclonal antibody, directed against a determinant in the COOH-terminal domain, allows localization of two identical determinants at or near the end of the subunit stalk. The same antibody was used to place two additional determinants at the periphery of stalkless subunits, in an area from which a stalk might be expected to project. A second antibody, directed against an epitope in the amino-terminal portion of L7/L12, caused loss of stalks from the 50 S subunits. The micrographs showed symmetrical oligometric complexes of the dissociated dimeric protein with bivalent antibody. Antibodies were also seen to bind to the body of stalkless subunits, in a region near the COOH-terminal sites. The results are explained by a model in which one dimer of protein L7/L12 exists in a folded conformation on the subunit body and the second dimer occurs in an extended conformation in the subunit stalk.