Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.     

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.     

Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site

Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl2. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by [3H] N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.     

Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.     

Insulin suppresses hepatitis B surface antigen expression in human hepatoma cells

The human hepatoma Hep3B cells contain integrated hepatitis B viral genome and continually secret hepatitis B surface antigen (HBsAg). The production of HBsAg (but not alpha-fetoprotein) was suppressed by addition of low concentrations (0.1-1 nM) of insulin into serum-free medium. In addition, the suppression of HBsAg production by insulin was paralleled with the decrease in HBsAg mRNA abundance. Insulin also cause a rapid rate of disappearance of HBsAg mRNA (t 1/2, 2 h) in Hep3B cells. The Hep3B cells carry specific receptor with high affinity for insulin (Kd = 1.8 nM). The receptor showed an insulin-dependent protein tyrosine kinase activity. The half-maximal insulin concentration for the activation of the receptor kinase was about 5 nM. Only very high concentrations of insulin-like growth factor I and human proinsulin can compete for the insulin receptor binding and suppress HBsAg production, this suggests that insulin may act through its receptor binding to suppress HBsAg expression in human hepatoma Hep3B cells.     

Enhanced secretion of Escherichia coli beta-lactamase by a spontaneous erythromycin-resistant mutant of Bacillus subtilis

An extracellular-protease-deficient mutant, ME142, was isolated from Bacillus subtilis as a spontaneous erythromycin-resistant (Eryr) clone. This mutant showed conditional sporulation and only sporulated normally in the absence of erythromycin. In the presence of the antibiotic, sporulation was greatly reduced. Production of extracellular proteases by ME142 also exhibited conditional deficiency, possibly due to pleiotropic effects of the sporulation deficiency. The production of protease was 2-10% that of the wild-type level in the presence of erythromycin. ME142 showed poor competence for transformation even in the absence of erythromycin; however, derivatives of ME142 were isolated which had the same Eryr phenotype but which exhibited normal competence. One such mutant, ME162, was used as a host for the secretion of Escherichia coli beta-lactamase. The amount of beta-lactamase in the culture supernatants of ME162 increased significantly when the cells were cultured with erythromycin, suggesting that proteolysis of the beta-lactamase in the supernatants of ME162 was greatly reduced as compared to that in the supernatants of the wild-type strain.     

A recessive mutation causing imperforate vagina in mice

A recessive mutation (ipv) causing imperforate vagina was discovered in a line of mice selected for low lean tissue mass as a proportion of body weight. Two full sisters were found to have marked swelling of the perineum and complete closure of the vagina. Crosses of heterozygotes identified by progeny testing produced a female progeny ratio not different from the 3 normal: 1 affected (chi 2 = 0.695; p less than .3) expected on the basis of a recessive allele at a single autosomal locus. As a consequence of the imperforate vagina, the uterus and vagina were greatly distended by fluid. The uterus of affected females displayed a swollen uterine lumen and thin endometrial stroma and muscularis. Ovarian tissue of affected females was similar to that of normal mice, and affected females produced ova that were normal in appearance. The mutation causing an imperforate vagina may present a useful model for studying the basis of abnormal vaginal development in other species and increasing the understanding of normal vaginal development in the mouse.     

Inhibitory effect of anti-class II antibody on the spontaneous activation of B cells in patients with systemic lupus erythematosus. Analysis with IL-1 production and IL-1 receptor expression

The mechanism of the spontaneous activation of B cells in patients with SLE was analyzed from the standpoint of the production of IL-1 from B cells and the expression of IL-1R on B cells. SLE B cells spontaneously produced IL-1-like factors which stimulated murine thymocyte proliferative responses. Their m.w. was about 17,000 and their isoelectric point was 4.8. The IL-1-like activity produced by B cells was absorbed with rabbit anti-IL-1 alpha antibody, but not with anti-IL-1 beta antibody. The differentiation of SLE B cells was enhanced by rIL-1 alpha, beta or IL-1-like factors produced by SLE B cells in a concentration-dependent manner. SLE B cells expressed large number of IL-1R detected by FITC-conjugated IL-1 alpha. By a Percoll gradient density centrifugation, IL-1-producing cells and B cells responsive to IL-1 were enriched in a higher density fraction, but were reduced in a lower density fraction. IL-1R-positive B cells were enriched in the lower density fraction, but were depleted in the higher density fraction. However, the expression of IL-1R on the lower density B cells was reduced by 2-day culture. The expression of IL-1R on the higher density B cells was increased during a 2-day culture. Anti-class II antibody inhibited the production of IL-1R on the higher density B cells. These results suggest that the cellular interaction among B precursor cells mediated by class II Ag induces the production of IL-1 and the expression of its receptors on their surface and the interaction between IL-1 and its receptors stimulates B precursor cells to spontaneously differentiate into Ig-producing cells as an autocrine mechanism in patients with SLE.     

Dual role of the CD44 molecule in T cell adhesion and activation

Studies of T cell adhesion and activation reveal two new functions of the CD44 molecule, a molecule now recognized to be identical to three molecules of functional interest: Pgp-1, Hermes, and extracellular matrix receptor type III (ECMRIII). By screening for mAb which inhibit T cell adhesion to E, we have identified a functionally unique CD44-specific mAb, NIH44-1, which partially inhibits T cell rosetting by binding to CD44 on the E. NIH44-1, which immunoprecipitates a protein of 85 to 110 kDa with broad tissue distribution, was determined to be specific for CD44 based on comparison of its tissue distribution with multiple CD44-specific reference mAb and sequential immunoprecipitation with such mAb. Anticipating a role for many adhesion molecules in signal transduction, we studied the effect of CD44 mAb on T cell activation and observed that CD44 mAb dramatically augments T cell proliferation induced by CD3- and CD2-receptor-mediated activation. The augmentation of the response to immobilized CD3 mAb by exhaustively monocyte-depleted T cells indicates that augmentation can be mediated by binding to the T cell. Thus, our studies demonstrate specific new roles for CD44 in T cell adhesion and activation. Furthermore, we suggest that: 1) CD44 has a role in adhesion of cells of multiple lineages; and 2) CD44 may participate in adhesion not (only) by functioning as an adhesion receptor but rather by serving as an anchorage site for other adhesion molecules.     

An electrophoretic polymorphism in salivary amylases (Amy-1) of mastomys (Praomys coucha).

An electrophoretic polymorphism of salivary amylases (Amy-1) in mastomys (Praomys coucha) (MWC, MRJ and MCC strains) was detected. Amylase in MWC or MRJ saliva, which migrated fast toward the anode, was designated as AMY-1A, and that in MCC saliva migrating slowly as AMY-1B. Salivary amylases are controlled by a pair of codominant alleles at a single autosomal locus (Amy-1). No polymorphism was seen in pancreatic amylases (Amy-2). The frequencies of these phenotypes did not differ between the sexes. Some isoamylases were observed and these were different from those in mouse or rat.