Copper(II) (3,5-diisopropylsalicylate)2 oxidizes thiols to symmetrical disulfides and oxidatively converts mixtures of 5-thio-2-nitrobenzoic acid and nonsymmetrical 5-thio-2-nitrobenzoic acid disulfides to symmetrical disulfides

L-cysteine, D-penicillamine, and L-glutathione were oxidized to symmetrical disulfides in the presence of Cu(II)(3,5-DIPS)2 and air-oxygen at physiologic pH, 7.3. Air-oxygen caused the oxidation of thiol reduced copper, Cu(I), to Cu(II), as evidenced by expected spectrophotometric changes in these reaction mixtures. L-cysteine, D-penicillamine, and L-glutathione formed mixed disulfides and TNB with the addition of DTNB to solutions of these thiols. The observed order of reactivity for these thiols with DTNB was: L-cysteine greater than D-penicillamine greater than L-glutathione. Surprisingly, Cu(II)(3,5-DIPS)2 converted these mixed disulfides to their symmetrical disulfides and DTNB, and although the initial conversion rate was rapid, complete conversion required more than two hours. These observations suggest caution with regard to the spectrophotometric determination of thiols immediately after the addition of Ellman's reagent. These results also clarify an earlier report concerning the oxidation of thiols by Cu(II)(o-phenanthroline)2 and offer caution with regard to the determination of thiols using DTNB in the presence of copper complexes. Spectrophotometric data are provided in support of the suggestion that analysis of plasma or cellular samples for thiols be done in the absence of copper(II) complexes to avoid false negative results.     

Tubulin structure and biochemistry.

In the past year, much has been learned about structure-function correlations in the tubulin molecule, and specifically about the nature and roles of post-translational modifications and tubulin isotypes. The interactions between tubulin and its ligands--both microtubule-associated proteins and anti-mitotic drugs--are becoming clearer at the molecular level.     

Comparison of the carbohydrate composition of rat and human corticosteroid-binding globulin: species specific glycosylation.

We have examined the carbohydrate composition of corticosteroid-binding globulin (CBG) obtained from rat and human serum. Rat CBG contained a carbohydrate composition that was strikingly different from that of human CBG. Like other glycoproteins that circulate in human plasma, human CBG had a carbohydrate composition that was consistent with the presence of biantennary and triantennary oligosaccharide structures. In contrast, the carbohydrate composition of rat CBG indicated the presence of more than one sialic acid residue per antenna. It is not clear whether rat CBG contains a carbohydrate structure with sialic acids attached to both galactose and N-acetylglucosamine on the same antenna, or a terminal disialylated structure (sialic acid linked alpha 2-8 to sialic acid). These structural variations may play a role in the interaction of CBG with its receptor.     

The molecular mechanism of inhibition of alpha-type DNA polymerases by N2-(butylphenyl)dGTP and 2-(butylanilino)dATP: variation in susceptibility to polymerization.

Calf thymus DNA polymerase alpha (pol alpha) and bacteriophage T4 DNA polymerase (pol T4) were exploited as model enzymes to investigate the molecular mechanism of inhibitory action of N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butyl-anilino)dATP (BuAdATP) on the BuPdNTP-susceptible alpha polymerase family. Kinetic analysis of inhibition of pol alpha with mixtures of complementary and noncomplementary template:primers indicated that both nucleotides induced the formation of a polymerase: inhibitor:primer-template complex. Primer extension experiments using the guanine form as the model analog indicated that pol alpha cannot utilize these nucleotides to extend primer termini. In contrast, pol T4 polymerized BuPdGTP, indicating that resistance to polymerization is not a common feature of the inhibitor mechanism among the broad membership of the alpha polymerase family.     

Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.     

The effects of fetal exposure to 1,2-dibromo-3-chloropropane on adult male reproductive function

Several drugs have been shown to cross the placental barrier and affect the fetal testis causing a reduction in testosterone with a resultant impairment of sexual differentiation and an ultimate problem in adult sexual function. In this study, pregnant female rats were treated with 25 mg/kg of the pesticide 1,2-dibromo-3-chloropropane (DBCP). Treatment began on Days 14.5, 16.5, or 18.5 and continued through Day 19.5 of gestation. Some animals were killed on Day 20.5 of intrauterine life and fetal intratesticular testosterone was measured. All other animals were allowed to deliver, and the males were raised to adulthood. At adulthood, body, testis, prostate glands and seminal vesicle weights were recorded. Intratesticular testosterone and luteinizing hormone (LH) receptors were measured. Male and female sexual behavior was quantified and the volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus was calculated. The histological appearance of the testis was also examined. Treatment for 6 days during fetal life with DBCP decreased intratesticular testosterone by 50% compared to controls at 20.5 days of gestation. At adulthood, all male rats treated during fetal life had a reduced body weight that was correlated with the duration of exposure. Adult testis weight was reduced to 75% of controls as a result of 2 days of fetal exposure to DBCP, whereas 4 and 6 days of exposure during fetal life reduced testis weight by greater than 90%. LH receptors and intratesticular testosterone, in the adults treated during fetal life, were also dramatically reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural changes in the NH2-terminal domain of fibronectin upon interaction with heparin. Relationship to matrix-driven translocation

The effects of heparin and various related polysaccharides on the circular dichroic spectra of fibronectin and its 31-kDa NH2-terminal tryptic fragment were studied. These effects were evaluated with respect to (i) spectral features of the native proteins that are sensitive to pH denaturation and breaking of disulfide bonds, (ii) sensitivity of spectral changes to Ca2+, and (iii) the fibronectin-dependent interfacial interaction known as "matrix-driven translocation." We found that native heparin causes an attenuation of the positive CD peak at 228 nm with both the intact protein and the fragment, and causes a small but reproducible red shift in the spectrum of the fragment. All of these changes are analogous to spectral changes seen with denaturation or reduction of the proteins. In contrast to the situation with the intact protein, the heparin-induced spectral changes in the fragment were abolished in the presence of 10 mM Ca2+. Desulfation of heparin lessened or destroyed its ability to induce these changes, and carboxymethylated heparin and dextran sulfate induced different kinds of spectral alterations. Fibronectin and heparin determinants required for the induction of the characteristic spectral shift of the NH2-terminal domain corresponded to those required for matrix-driven translocation, suggesting that the associated conformational change in fibronectin plays a role in this biophysical effect.     

Native-like intermediate on the folding pathway of Escherichia coli succinyl-CoA synthetase

The transition between the native and denatured states of the tetrameric succinyl-CoA synthetase from Escherichia coli has been investigated by circular dichroism, fluorescence spectroscopy, cross-linking by glutaraldehyde and activity measurements. At pH 7.4 and 25 degrees C, both denaturation of succinyl-CoA synthetase by guanidine hydrochloride and refolding of the denatured enzyme have been characterized as reversible reactions. In the presence of its substrate ATP, the denatured enzyme could be successfully reconstituted into the active enzyme with a yield of 71-100%. Kinetically, reacquisition of secondary structure by the denatured enzyme was rapid and occurred within 1 min after refolding was initiated. On the other hand, its reactivation was a slow process which continued up to 25 min before 90% of the native activity could be restored. Both secondary and quaternary structures of the enzyme, reconstituted in the absence of ATP, were indistinguishable from those of the native enzyme but the renatured protein was catalytically inactive. This observation indicates the presence of catalytically inactive tetramer as an intermediate in the reconstitution process. The reconstituted protein could be reactivated by ATP even 10 min after the reacquisition of the native secondary structure by the refolding protein. However, reactivation of the protein by ATP 60 min after the regain of secondary structure was significantly less, suggesting that rapid refolding and reassociation of the monomers into a native-like tetramer and reactivation of the tetramer are sequential events; the latter involving slow and small conformational rearrangements in the refolded enzyme that are likely to be associated with phosphorylation.     

Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin

Receptor-mediated endocytosis of 125I-insulin and 125I-prolactin into liver parenchymal cells has been studied by quantitative subcellular fractionation. Differential centrifugation yielded three particulate fractions, N (nuclear), ML (large granule), and P (microsomes), and a final supernatant (S). Quantitative differences in the extent and rates of accumulation of 125I-insulin and 125I-prolactin into the fractions were observed. The acidotropic agent chloroquine and the microtubule disrupting agent colchicine were administered separately to rats. The agents increased significantly the T 1/2 of hormone clearance from the liver and augmented the accumulation of both ligands in the low-speed ML fraction. However, differences in the rates of accumulation of insulin and prolactin into all cell fractions were still maintained. Analytical centrifugation of each of the particulate fractions was carried out in order to determine if different endocytic components were specific to insulin or prolactin internalization. This was not the case. An "early" endosomal component of density 1.11 was identified in microsomes. A "late" endosome of density 1.10 was identified in the large granule (ML) fraction. Both endosomal components appeared to accumulate insulin and prolactin but at different rates. Marker enzyme analysis identified the presumed plasma membrane component in microsomes (density approximately 1.155). This component showed a significant difference in the rate of loss of 125I-insulin (T 1/2 approximately 4.1 min) as compared to that of 125I-prolactin (T 1/2 approximately 12.7 min). A further difference in the handling of the ligands was observed in early endosomes.(ABSTRACT TRUNCATED AT 250 WORDS)