The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins.

Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with lipase as is native colipase and that exhibits a strong emission band at 550 nm. Addition of micellar concentrations of taurodeoxycholate causes a 4.3-fold increase in the emission maximum as well as a 70 nm blue shift to 480 nm. Inclusion of oleic acid to form a mixed micelle reduces these spectral effects. Scatchard analysis of the data yield a Kd of 6.8 X 10(-4) M and a single colipase-binding site for taurodeoxycholate micelles. The data, by analogy to a phospholipase system, are consistent with a direct insertion of dansyl-NH-tyrosine-55 into the micelle. The presence of a single tryptophan residue (Trp-52) in equine colipase provides an intrinsic fluorescent probe for studying protein-micelle interaction. The emission maximum of horse colipase at 345 nm indicates a solvent-accessible tryptophan residue which becomes less so on binding of micelles. A blue shift of 8 nm and a 2-fold increase in amplitude is indicative of a more hydrophobic environment for tryptophan induced by taurodeoxycholate micelles. There is also a decrease in KSV for acrylamide quenching in the presence of micelles, which further supports a loss of solvent accessibility. The most dramatic pH effects are observed with KI quenching, and may indicate the presence of negative charges near Trp-52.     

Participation of the lone tryptophan residue of rat alpha-foetoprotein in its drug-binding sites. Comparison with rat serum albumin.

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.     

Binding of NAD+ by cholera toxin.

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.     

Phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) influence mushroom tyrosinase activity.

A series of phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) were synthesized in order to study their interaction with mushroom tyrosinase. 1-Amino-2-(3,4-dihydroxyphenyl)ethylphosphonic acid and 1-amino-2-(3,4-dimethoxyphenyl)ethylphosphonic acid turned out to be substrates for mushroom tyrosinase with Km values of 3.3 mM and 9.3 mM respectively. Shortening of the alkyl chain by one methylene group gave amino-(3,4-dihydroxyphenyl)methylphosphonic acid, one of the most powerful known inhibitors of this enzyme. This compound, racemic as well as in its optically active forms, exerts a mixed type of inhibition with an affinity for the enzyme one order of magnitude greater than that of the natural substrate.     

Characterization and physiological function of glutathione reductase in Euglena gracilis z.

The purified glutathione reductase was homogeneous on polyacrylamide-gel electrophoresis. It had an Mr of 79,000 and consisted of two subunits with a Mr of 40,000. The activity was maximum at pH 8.2 and 52 degrees C. It was specific for NADPH but not for NADH as the electron donor; the reverse reaction was not observed. The Km values for NADPH and GSSG were 14 and 55 microM respectively. The enzyme activity was markedly inhibited by thiol inhibitors and metal ions such as Hg2+, Cu2+ and Zn2+. Euglena cells contained total glutathione at millimolar concentration. GSH constituted more than 80% of total glutathione in Euglena under various growth conditions. Glutathione reductase was located solely in cytosol, as were L-ascorbate peroxidase and dehydroascorbate reductase, which constitute the oxidation-reduction cycle of L-ascorbate [Shigeoka et al. (1980) Biochem. J. 186, 377-380]. These results indicate that glutathione reductase functions to maintain glutathione in the reduced form and to accelerate the oxidation-reduction of L-ascorbate, which scavenges peroxides generated in Euglena cells.     

Transformation and rescue of a flightless Drosophila tropomyosin mutant.

In the Drosophila flightless mutant Ifm(3)3, a transposable element inserted into the alternatively spliced fourth exon of the tropomyosin I (TmI) gene prevents proper expression of Ifm-TmI, the tropomyosin isoform found in indirect flight muscle. We have rescued the flightless phenotype of Ifm(3)3 flies using P-element-mediated transformation with a segment of the Drosophila genome containing the wild-type TmI gene plus 2.5 kb of 5' flanking and 2 kb of 3' flanking DNA. The inserted TmI gene is expressed with the proper developmental and tissue specificity, although its level of expression varies among the five transformed lines examined. These conclusions are based on analyses of flight, myofibrillar morphology, and TmI RNA and protein levels. A minimum of two copies of the inserted TmI gene per cell is necessary to restore flight to most of the flies in each line. We also show that the Ifm-TmI isoform is expressed in the leg muscle of wild-type flies and is decreased in Ifm(3)3 leg muscle. Homozygous Ifm(3)3 mutants do not jump. The ability to jump can be restored with a single copy of the wild-type TmI gene per cell.     

High-affinity binding sites involved in the import of porin into mitochondria.

The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane.     

The regulation of glutamine and ketone-body metabolism in the small intestine of the long-term (40-day) streptozotocin-diabetic rat.

The small intestine is the major site of glutamine utilization in the mammalian body. During prolonged (40-day) streptozotocin-diabetes in the rat there is a marked increase in both the size and the phosphate-activated glutaminase activity of the small intestine. Despite this increased capacity, intestinal glutamine utilization ceases in diabetic rats. Mean arterial glutamine concentration fell by more than 50% in diabetic rats, suggesting that substrate availability is responsible for the decrease in intestinal glutamine use. When arterial glutamine concentrations in diabetic rats were elevated by infusion of glutamine solutions, glutamine uptake across the portal-drained viscera was observed. The effect of other respiratory fuels on intestinal glutamine metabolism was examined. Infusions of ketone bodies did not affect glutamine use by the portal-drained viscera of non-diabetic rats. Prolonged diabetes had no effect on the activity of 3-oxoacid CoA-transferase in the small intestine or on the rate of ketone-body utilization in isolated enterocytes. Glutamine (2 mM) utilization was decreased in enterocytes isolated from diabetic rats as compared with those from control animals. However, glutaminase activity in homogenates of enterocytes was unchanged by diabetes. In enterocytes isolated from diabetic rats the addition of ketone bodies or octanoate decreased glutamine use. It is proposed that during prolonged diabetes ketone bodies, and possibly fatty acids, replace glutamine as the major respiratory fuel of the small intestine.     

Studies on the interactions of human pancreatic elastase 2 with human alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin.

Several intermediates in the reaction of 2-methylglutamate with glutamate decarboxylase from Escherichia coli were detected by stopped-flow spectrophotometry and by rapid-scanning spectrophotometry after conventional mixing. Structures were assigned to intermediates on the basis of kinetic and spectral evidence. In the early stages of the reaction an intermediate with the properties expected of a geminal diamine accumulated significantly. Changes consistent with the conversion of this species into the external aldimine were also observed. The course of product formation was determined and linked with spectral changes taking place in the bound coenzyme. The effect of the minor decarboxylation-dependent transamination that accompanies the major reaction was analysed.