Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59

It has been proposed that the unliganded nontransformed form of steroid hormone receptor is a heterooligomer comprising, in addition to the hormone-binding subunit, two associated proteins: a heat shock protein of MW 90,000 (hsp90) and another protein of MW 59,000 (p59). Using monoclonal antibodies, we demonstrate immunocytochemically the presence of both hsp90 and p59 in cell nuclei of progesterone target cells of the rabbit uterus. While steroid receptors (e.g., progesterone receptors) appear to be exclusively nuclear, we find p59 predominantly in the cell nuclei and hsp90 in both the nucleus and the cytoplasm. In addition, Western blotting of high-salt extracts of nuclear proteins detects the presence of hsp90 and p59 in the nuclei of rabbit uterus. These observations are consistent with the presence of the untransformed heterooligomeric form of steroid hormone receptors in the nuclei of target cells.     

Processing of Ada protein by two serine endoproteases Do and So from Escherichia coli

Two soluble serine proteases Do and So from Escherichia coli were found to distinctively cleave the purified, 39 kDa Ada protein into fragments with sizes of 12-31 kDa. Protease So appears to generate a C-terminal 19 kDa polypeptide, similarly to OmpT protease. In addition, the purified 19 kDa C-terminal half of Ada protein can be further processed mainly to an 18 kDa fragment by protease So and to a 12 kDa by protease Do. These results suggest that proteases Do and So are involved in endogenous cleavage of Ada protein, which may play a role in down-regulating the adaptive response to alkylating agents.     

Quaternary structure of pigeon liver malic enzyme

Pigeon liver malic enzyme (EC 1.1.1.40) has a double dimer quaternary structure. The NADP+ analogs, aminopyridine adenine dinucleotide phosphate and nicotinamide-1,N6-ethenoadenosine dinucleotide phosphate, bind to the enzyme anti-cooperatively. In the presence of non-cooperative competing ligand NADP+, the binding parameter Hill coefficients of these analogues changed very little. Binding of L-malate with enzyme-AADP+ complex first enhanced then reduced the nucleotide fluorescence. Two L-malate binding sites, with Kd values of 23-30 and 270-400 microM, respectively. for the tight and weak binding sites were postulated. A hybrid model between the sequential and pre-existing asymmetrical models was proposed for the pigeon liver malic enzyme.     

The distribution and localization of the stage-specific GP31 antigen from infective Ostertagia circumcincta larvae.

The 31,000 mol. wt glycoprotein (GP31) antigen of infective third-stage (L3) Ostertagia circumcincta larvae was shown, by surface labelling experiments and immunofluorescent antibody staining of whole larvae and larval sections, to be distributed internally. When transverse sections of L3 O. circumcincta, taken from the anterior pharyngeal region, were further examined by electron microscopy, after immunogold staining with rabbit anti-GP31 antiserum, the GP31 antigen was found to be specifically located in 'secretory organelles' within the cells of the oesophageal glands. By in vitro culturing L3 O. circumcincta in medium supplemented with 35S-methionine and then analysing the excretory-secretory material released by the larvae, it was found that the GP31 molecule was one of the major components of the excretory-secretory complex. The purified GP31 molecule had no detectable proteolytic activity in protein degradation assays. On examination of Triton X-100 extracts of infective larvae from other nematode parasite species, a predominant antigen similar to GP31 was found in Trichostrongylus colubriformis and Haemonchus contortus, but in Toxocara canis a minor component corresponding in mol. wt to GP31 was also detected. Based on these results the possible role of GP31 as a candidate antigen for a broad spectrum molecular vaccine against gastrointestinal nematode parasites in sheep is discussed.     

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.     

The role of carbohydrate moieties of cholecystokinin receptors in cholecystokinin octapeptide binding: alteration of binding data by specific lectins

Cholecystokinin (CCK) receptors on rat pancreatic acini have been demonstrated to be glycoproteins. In order to study whether their carbohydrate moieties play a role in ligand binding, membrane preparations (adjusted to 0.2 mg protein me) were incubated with 20 pM 125-I-CCK octapeptide (125I-CCK8) for 4 h at 30 degrees C in the presence of lectins with different sugar specificities. Concanavalin A, soy-bean agglutinin, and peanut agglutinin in concentrations up to 1 mM did not alter specific 125I-CCK8 binding. Ulex europeus lectin I showed a dose-dependent enhancement of CCK binding up to 150% of controls at a concentration of 1 mM. Wheat-germ agglutinin (WGA) was the only lectin found to have an inhibitory effect. Inhibition was dose-dependent, with maximal reduction attained at 42 nM, but CCK binding was only partially inhibited to 66.2 +/- 4.4%. Inhibition by WGA was prevented by the presence of N-acetyl-D-glucosamine or N,N',N"-triacetylchitotriose, sugars that are specific for WGA. The inhibitory effect of WGA was not due to an increase in non-specific binding, increased CCK degradation, or CCK binding to WGA. Binding data indicated that the presence of WGA resulted in a decrease in receptor affinity (Kd = 567 +/- 191 v. 299 +/- 50 pM). No significant change in the number of available binding sites was observed. This suggests that WGA is not binding to the active binding site. It is conceivable that binding of WGA to N-acetyl-D-glucosamine or its polymers can lead to a conformational change in the receptor protein, and that this carbohydrate moiety is essential for optimal receptor-ligand interaction.     

Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin

The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.     

Different molecular sizes for Na(+)-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesicles

We compared several features of Na(+)-dependent phosphono[14C]formic acid (PFA) binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles. From kinetic analyses, we estimated an apparent Km for PFA binding of 0.86 mM, an order of magnitude greater than that for phosphate and the high-affinity phosphate transport system. A hyperbolic Na(+)-saturation curve for PFA binding and a sigmoidal Na(+)-saturation curve for phosphate transport were demonstrated; based on these data, we estimated stoichiometries of 1:1 for Na+/PFA and 2:1 for Na+/phosphate. By radiation inactivation analysis, target sizes for brush border membrane protein(s) mediating Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport corresponded to molecular masses of 555 +/- 32 kDa and 205 +/- 36 kDa, respectively. Similar analysis of the phosphate-inhibitable component of Na(+)-dependent PFA binding gave a target size of 130 +/- 28 kDa. We also demonstrated that phosphate deprivation, which elicits a 2.6-fold increase in brush border membrane Na(+)-dependent phosphate transport, had no effect on either Na(+)-dependent PFA binding or on the target size for PFA binding. However, phosphate deprivation appeared to increase the target size for phosphate transport (from 255 +/- 32 to 335 +/- 75 kDa (P less than 0.01]. In summary, we present evidence for several differences between Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles and suggest that PFA may not interact exclusively with the proteins mediating Na(+)-phosphate co-transport.