A new substrate for porcine pepsin possessing cryptic fluorescence properties

A fluorescent substrate for porcine pepsin, 50-dimethylaminonaphthalene-1-sulfonyl (Dns)-Ala-Ala-Phe-Phe-3-[4-(N-CH3)-pyridyl]propyl-1-oxy ester has been synthesized. It is stable, soluble from pH 1 to 7, and is readily hydrolyzed by pepsin with values of 288 (+/- 40) s-1 for kcat, 0.039 mM (+/- 0.005) for Km, and 7510 s-1 mM-1 (+/- 500) for kcat/Km in sodium formate, pH 3.1. Kinetic studies were carried out by following the increased fluorescence (300-nm excitation, 525-nm emission) as hydrolysis occurred. The products of hydrolysis were identified and established that the peptide bond between the phenylalanine residues is cleaved by pepsin. The inhibition of pepsin catalysis by pepsinogen (1-12) activation peptide was studied in order to compare the inhibition of the reaction of pepsin with Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ with that obtained by the standard milk-clotting assay. The inhibition results were comparable. Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ should be a valuable tool for studies of pepsin because of its solubility over an extended pH range, its excellent turnover rate, and the ease with which the hydrolysis can be followed.     

alpha-chloroepoxides. 2. Mutagenicity of 1-chlorocyclohexene oxide and its thermal isomerization product, 2-chlorocyclohexanone

The pseudo-first-order hydrolysis rate constants in pH 7.4 buffer-acetone solution are reported for 1-chlorocyclohexene oxide and 2-chlorocyclohexanone at 0, 25 and 37 degrees C. The rate constants, in conjunction with product studies, demonstrate that the hydrolysis of 1-chlorocyclohexane oxide quantitatively affords 2-hydroxycyclohexanone and that there is no significant isomerization of 1-chlorocyclohexene oxide to 2-chlorocyclohexanone during the hydrolysis. Both 1-chlorocyclohexene oxide and 2-chlorocyclohexanone were reacted with 2-aminopyridine, a model for adenine, to yield the same product, N-(2'-pyridyl)-2-aminocyclohexanone. The mutagenicity of 1-chlorocyclohexene oxide and 2-chlorocyclohexanone in the Ames liquid incubation assay using TA100 shows 2-chlorocyclohexanone to be slightly more active than 1-chlorocyclohexene oxide, in spite of the finding that 1-chlorocyclohexene oxide is clearly a more reactive electrophile than 2-chlorocyclohexanone. These results are interpreted to indicate the important role that hydrolysis (detoxification) plays in the in vitro evaluation of the proposed ultimate electrophilic metabolites of chloroolefins.     

Cystic fibrosis: present status and future prospects in detection of patients and carriers.

Development of a sensitive, easily performed, reliable test would be an important advance in detecting cystic fibrosis, improving genetic counselling and providing early effective treatment. The sweat chloride test, which is reliable in diagnosis, is technically too difficult for a screening program, and only reliably detects homozygotes. In contrast, the meconium test for detecting homozygote newborns is simple, inexpensive, reasonably specific but its general application has yet to be evaluated. Detection of serum components is the basis of two new tests to distinguish patients with cystic fibrosis and carriers. The effect of these serum components on ciliary activity is the principle of one test, an extremely difficult procedure that is subjective and lacks sufficient specificity for routine use. The second test, in which serum components are separated by isoelectric focusing, may provide an objective biochemical means of detecting both homozygotes and heterozygotes.     

Fluorescent alamethicin fragments A study of membrane activity and aqueous phase aggregation

The linear polypeptide antibiotic alamethicin is known to form channels in artificial lipid membranes. Synthetic 13- and 17-residue alamethicin fragments, labelled with a fluorescent dansyl group at the N-terminus, have been shown to translocate divalent cations across phospholipid membranes and to uncouple oxidative phosphorylation in rat liver mitochondria, in a manner analogous to the parent peptides. From studies of the aqueous phase aggregation behavior of the peptides, as well as their interaction with rat liver mitochondria, it is concluded that the interaction of the peptides with membranes is a complex process, probably involving both aqueous and membrane phase aggregation.     

Synthesis of carbamyl and ether analogs of phosphatidylcholines

A complete synthesis of 1-O-hexadecyl-2-O-N-(heptadec-8-cis-enyl)carbamyl-sn-glycero-3-Phosphocholine, a novel analog of phosphatidylcholine, has been described. Each step is simple to perform and gives the desired products in high yield. Also, some of the intermediates formed during the synthesis have been efficiently utilized to prepare 1-O-hexadecyl-2-O-oleyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphochloine and 3-O-hexadecyl-2-oeloyl-sn-glycero-1-phosphocholine. These phosphatidylcholine (PC) analogs are useful for studying the possible role of phospholipases in the capture and lyses of liposomes in vivo.     

Blood group i and I activities of "lacto-N-norhexaosylceramide" and its analogues: the structural requirements for i-specificities.

A pure straight chain ceramide hexasaccharide (“lacto-N-norhexaosylceramide” Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glc→Ceramide) showed strong i-activity determined by hemagglutination inhibition and by radioimmunoassay with five out of six anti-i antisera. Two repeating Galβ1→4GlcNAc residues and GlcNAcβ1→3Gal residues could be essential for the full expression of this activity; eleven closely related analogues including those derived by chemical modification had lower or no detectable activity. The same structure reacted also with some anti-I antisera. The strong i-activity and the moderate I-activity were both abolished by elimination of the terminal Gal or by removal of the N-acetyl groups of the two GlcNAc residues.     

Two-step aminoacylation of tRNA without channeling in Archaea

Catalysis of sequential reactions is often envisaged to occur by channeling of substrate between enzyme active sites without release into bulk solvent. However, while there are compelling physiological rationales for direct substrate transfer, proper experimental support for the hypothesis is often lacking, particularly for metabolic pathways involving RNA. Here, we apply transient kinetics approaches developed to study channeling in bienzyme complexes to an archaeal protein synthesis pathway featuring the misaminoacylated tRNA intermediate Glu-tRNA^Gln. Experimental and computational elucidation of a kinetic and thermodynamic framework for two-step cognate Gln-tRNA^Gln synthesis demonstrates that the misacylating aminoacyl-tRNA synthetase (GluRS^ND) and the tRNA-dependent amidotransferase (GatDE) function sequentially without channeling. Instead, rapid processing of the misacylated tRNA intermediate by GatDE and preferential elongation factor binding to the cognate Gln-tRNA^Gln together permit accurate protein synthesis without formation of a binary protein-protein complex between GluRS^ND and GatDE. These findings establish an alternate paradigm for protein quality control via two-step pathways for cognate aminoacyl-tRNA formation.     

Thermostability of α-mannosidase in plasma from cystic fibrosis patients and carriers

The hypothesis presented is that the different classes of c-type cytochrome originated as proteins located in the bacterial periplasmic space, or on the periplasmic side of the cytoplasmic membrane. In these locations, covalent bonds between haem and protein prevented the haem from being lost to the surrounding medium. Subsequent evolution has led to internal location of c-type cytochromes in eucaryotes and cyanobacteria. The covalent links have been retained because of their structural role; a b-type cytochrome could be created with similar molecular properties, but its formation would require a large evolutionary jump. If this hypothesis is correct, it should be useful in unravelling electron transport chains with unconventional donors or acceptors. Apparent exceptions deserve further investigation.     

Metabolism of 2,4',5-trichlorobiphenyl: role of the intestinal microflora in the formation of bronchial-seeking methyl sulphone metabolites in mice

Groups of germ-free and conventional mice were treated with 2,4',5-trichlorobiphenyl (triCB) and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. Conventional mice accumulated significantly more extractable radioactivity both in lung and kidney tissues when compared to germ-free mice. The extracted radioactivity in lung and kidney tissues was shown to be due to the accumulation of methyl-[35S]sulphonyl-triCB. The low radioactivity in lungs of the germ-free mice was also shown to be due to the accumulation of small amounts of the sulphones. The results indicate an involvement of the intestinal flora in the formation of methyl sulphone metabolites of triCB.     

The hapten structure of a developmentally regulated glycolipid antigen (SSEA-1) isolated from human erythrocytes and adenocarcinoma: a preliminary note

Glycolipid antigen reacting to the monoclonal antibody directed to the developmentally regulated antigen SSEA-1 was isolated from human erythrocytes and colonic adenocarcinoma. The antigens have the Le^x (Galβl→4[Fucα]→3]GlcNAcβl→R) or Le^y (Fucαl→2Galβl→4[Fucαl→3]GlcNAcβl→R) structure at the termini of the branched polylactosaminolipid. In addition, a novel polyfucosyl structure locating exclusively at the internal GlcNAc was detected in the tumor antigen. The antibody reacts with a simple monovalent Le^x glycolipid (Galβl→4[Fucαl→3]GlcNAcβl→3Galβl→4Glcβl→Cer) previously isolated from colonic carcinoma when presented at a high density on liposomes. The antibody therefore may react to the bivalent or multivalent Le^x or Le^y structure.     

A novel cyclic opioid peptide analog showing high preference for mu-receptors

The side-chain to side-chain cyclized opioid peptide analogs H-Tyr-D-Orn-Phe-Asp-NH2 (I) and H-Tyr-D-Lys-Phe-Glu-NH2 (II) were synthesized and tested in the guinea pig ileum and mouse vas deferens assays and in binding assays based on displacement of mu- and delta-opioid receptor-selective radioligands from rat brain membranes. The more rigid cyclic analog I containing a 13-membered ring structure showed very high preference for mu-receptors over delta-receptors, whereas the more flexible cyclic peptide II (15-membered ring) was non-selective. These results indicate that variation in the degree of conformational restriction of opioid peptides can produce drastic shifts in their receptor selectivity profile. Because of its high mu-receptor selectivity and rigidity cyclic analog I will be useful for determining the conformational requirements of mu-opioid receptors.     

Metabolism of β-alanyl-l-tyrosine in Sarcophaga bullata

Soluble enzymes have been demonstrated in extracts of Sarcophaga bullata larvae and pupae which both synthesize and hydrolyze the dipeptide β-alanyl-l-tyrosine (β-AT). In vitro assays for the synthesis and hydrolysis of β-AT were developed using thin-layer chromatography and (1-¹⁴C)β-alanine.The fat body was identified as the tissue origin of both the synthetase and hydrolase enzymes. The specific activity of β-AT synthetase in fat body extracts was constant throughout larval development, whereas hydrolytic activity in fat body extracts rose sharply after completion of the white puparium, reached a maximum in 12 to 18 hr, and then fell to its initial level. The rôle of the two enzymic activities in the formation of the puparium of S. bullata is discussed.     

Incorporation of exogenous glycosphingolipids in plasma membranes of cultured hamster cells and concurrent change of growth behavior

Globoside added to culture medium was taken up by NIL cells and accumulated as a component of plasma membrane. This was evidenced by the recovery of ³H-labelled globoside from plasma membrane fractions and by the higher chemical quantity of globoside found in NIL cells cultured in medium containing globoside. Concomitantly the following changes in growth behavior were manifested: a reduction in growth rate due to an extended prereplicative phase and a reduced saturation density which may result from changed adhesive properties of cells.     

In vivo metabolism of 3H-testosterone in adult male rats: effects of estrogen administration.

The metabolism of testosterone (T) was studied in normal adult male rats using a constant infusion of trace amounts of the 3H-steroid into a tail vein for 3 h in order to attain a state of equilibrium. Samples of plasma, liver, kidney, prostate, seminal vesicles and muscle were analysed for 3H-testosterone, 3H-5alpha-dihydrotestosterone (5alphaDHT) and 3H-5alpha-androstanediol (Adiol). When compared to the 3H-T level in plasma there were high levels of 3H-T in kidney and of 3H-5alphaDHT in prostate and seminal vesicles. Intraperitoneal estradiol valerate administration (100 mug/day) for 4 days decreased and 3H-5alphaDHT levels in the prostate and seminal vesicles. The estrogen administration increased the T metabolic clearance rate from 17.5 1/24 h/100 g body wt to 22.6 1/24 h/100 g body wt.     

Autolysis of membrane lipids in potato leaf homogenates: Effects of calmodulin and calmodulin antagonists

Potato leaves contain high levels of lipolytic acyl hydrolase activity which degrades phospholipids and galactolipids during homogenization and organelle isolation. Four calmodulin antagonists (dibucaine, tetracaine, trifluoperazine a and chlorpromazine) were found to inhibit the rate of hydrolysis of endogenous membrane lipids in homogenates of potato leaves. In contrast, the addition of calcium and purified calmodulin stimulated the rate of hydrolysis. These results indicate that a lipolytic acyl hydrolase activity in potato leaves appears to be mediated either directly or indirectly by calcium and calmodulin.     

Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines. Non-specific reactions with phenols and benzo[alpha]pyrene

Treatment of mutagenic primary aromatic amines with nitrous acid is known to decrease their mutagenicity. We examined some factors concerning the validity of using decreases in mutagenicity due to nitrous acid treatment as an indication of the presence of mutagenic primary aromatic amines in complex mixtures. We found that treatment of benzo[alpha]pyrene with nitrous acid for the extended periods of time previously employed leads to formation of three nitrobenzo[alpha]pyrene isomers. Some of the isomers are direct-acting mutagens for S. typhimurium with considerably greater mutagenicity than benzo[alpha]pyrene isomers. In attempts to minimize reaction of chemicals other than aromatic amines, we found that only very brief reaction periods are required for complete reaction of nitrous acid with representative aromatic amines, essentially eliminating their mutagenicity. During such brief reaction periods modification of benzo[alpha]pyrene is negligible, but phenols react readily. Chromatographic analysis indicated that reaction of nitrous acid with aromatic amines leads to the formation of families of products, thereby increasing the complexity of the mixtures in which the amines may occur. Thus, experiments examining the effects of nitrous acid on the mutagenic activity of complex mixtures must be carefully designed, and the results must be interpreted cautiously.