Purification and characterization of a large, tryptic fragment of human thyroid peroxidase with high catalytic activity

Thyroid peroxidase (TPO) was purified from human thyroid tissue, obtained at surgery from patients with Graves' disease, by a procedure similar to one that we had previously used for the purification of porcine TPO. The membrane-bound enzyme was solubilized by treatment of the thyroid particulate fraction with trypsin plus detergent. After precipitation with ammonium sulfate, the enzyme was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. Although a high degree of purification was achieved, the finally isolated product was considerably more heterogeneous than the TPO obtained from porcine thyroids. Several pools of active enzyme differing in values for A412/A280 and in specific activity were collected. Gel electrophoresis was performed under native, denaturing [sodium dodecyl sulfate (SDS)] and denaturing plus reducing conditions. Native gel electrophoresis indicated that the active enzyme (93 kDa) was heavily contaminated with an inactive 60-kDa fragment, which we were unable to remove by HPLC. The inactive fragment was highly antigenic when tested on immunoblots with an antibody to TPO. The presence of the inactive fragment greatly reduced values for A412/A280 in the finally purified human TPO. Two of the pools, with A412/A280 values of 0.159 and 0.273, were used for further testing. Catalytic activity was very similar in these two pools when measured on the basis of heme content by several different assays. Moreover, the specific activities of both, based on heme content, were very similar to those observed with a porcine TPO preparation with A412/A280 = 0.48. These findings indicate that the inactive 60-kDa fragment most likely did not contain heme. On SDS-polyacrylamide gel electrophoresis under reducing conditions, the 60-kDa fragment completely disappeared and was replaced by a 36- and a 24-kDa component. Amino terminal sequence information obtained on these components indicated that the 24-kDa component represents the amino terminal portion of the active 93-kDa fragment, whereas the 36-kDa fragment represents the carboxyl terminal portion. A model is proposed suggesting that the 60-kDa fragment was generated by trypsin cleavage of native TPO at two internal sites within a disulfide loop (res approximately 300 and res 564) and at one further internal site (res 280). In addition, trypsin cleavage is proposed at sites near the amino and carboxyl ends common to both the active 93-kDa and the inactive 60-kDa fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and localization of a cysteinyl residue critical for the trypsin-like catalytic activity of the proteasome.

Chemical modification of the proteasome with N-ethylmaleimide (NEM) was performed for the purpose of identifying amino acid residues that play a role in the enzyme's proteolytic function. Modification of the proteasome with NEM specifically and irreversibly suppressed one of the three peptidase activities of the enzyme, viz., the "trypsin-like" activity. Leupeptin, a reversible competitive inhibitor of this activity, protected the activity from NEM inactivation, suggesting that NEM modifies a residue in the leupeptin binding site. Comparisons of enzyme samples labeled with [14C]NEM either in the presence or in the absence of leupeptin allowed the identification of a proteasome subunit containing an NEM-modified, leupeptin-protected cysteinyl residue. The leupeptin protection experiments suggest that residues of this subunit contribute to the active site responsible for the proteasome's trypsin-like activity. This subunit was purified by reverse-phase high-performance liquid chromatography. Peptide mapping and N-terminal amino acid sequencing were employed to acquire information about the primary structure of the subunit, including the sequence surrounding the leupeptin-protected cysteinyl residue. The sequencing data suggest that this proteasome subunit is evolutionarily related to other proteasome subunits that have been sequenced, which show no homology to other known proteases. The assignment of a catalytic function to a member of the proteasome family supports the hypothesis that proteasome subunits represent a structurally and possibly mechanistically novel group of proteases.     

Newly assembled microtubules are concentrated in the proximal and distal regions of growing axons

We have investigated the sites of microtubule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the proportion of tyrosinated alpha-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain more tyr-tubulin than older (more long-lived) MTs. To quantify the relative proportion of tyr-tubulin in MTs, cultured rat sympathetic neurons were permeabilized under conditions that stabilize existing MTs and remove unassembled tubulin. The MTs were then double-stained with antibodies to tyr-tubulin (as a measure of the amount of tyr-tubulin in MTs) and to beta-tubulin (as a measure of total MT mass), using immunofluorescence procedures. Cells were imaged with a cooled charge-coupled device camera and the relative proportion of tyr-tubulin in the MTs was quantified by computing the ratio of the tyr-tubulin fluorescence to the beta-tubulin fluorescence using a novel application of digital image processing and analysis techniques. The amount of tyr-tubulin in the MTs was highest in the cell body and at the growth cone; peak ratios in these two regions were approximately 10-fold higher than for the axon shaft. Moving out from the cell body into the axon, the tyr-tubulin content declined over an average distance of 40 microns to reach a constant low value within the axon shaft and then rose again more distally, over an average distance of 110 microns, to reach a peak at the growth cone (average axon length = 358 microns). These observations indicate that newly assembled MTs are concentrated in the proximal and distal regions of growing axons and therefore that the cell body and growth cone are the most active sites of MT assembly dynamics in neurons that are actively extending axons.     

Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes

A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.     

Glutamate decarboxylase from barley embryos and roots. General properties and the occurrence of three enzymic forms.

Glutamate decarboxylase in extracts of barley has a Km value for L-glutamate of 22 mM and is activated by the addition of pyridoxal phosphate by up to 3.5 times. Sucrose-density-gradient experiments indicate the presence of two enzyme forms with molecular weights 256000 and 120000. The lower-molecular-weight form appears to be relatively inactive and spontaneously associates to the higher-molecular-weight form on storage. The enzyme is inhibited by thiol reagents and the distribution of activity on density gradients is altered in favour of the lower-molecular-weight form by the presence of 2-mercaptoethanol. After removal of the 2-mercaptoethanol spontaneous association to the higher-molecular-weight form occurs. The presence of oxygen in the extraction buffer and in the water during imbibition leads to a relative increase in the higher-molecular-weight form compared with situations where oxygen is excluded. In contrast, glutamate decarboxylase in extracts of 3-day-old barley roots has a Km value for L-glutamate of 3.1 mM and is activated up to 10% by addition of pyridoxal phosphate. The root enzyme occurs as a single species with molecular weight 310000 and this is unaffected by 2-mercaptoethanol although thiol reagents do act as weak inhibitors. The molecular weight is also unaffected by the presence or absence of oxygen in the extraction buffers.     

Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities

FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.