Pyroglutamyl N-termini of thermal polyamino acids.

It has been established indirectly that the N-termini of the thermal polyamino acids are pyroglutamic acid. This was determined by trifluoroacetic acid hydrolysis of the lactam ring followed by Dansyl labelling. The polyamino acids contained Ala, Gly, Glu, Leu, Phe, and Pro. In the experiments described here, the presence of pyroglutamic acid at the N-terminus of a polyamino acid was determined directly by the use of pyrrolidone carboxylyl peptidase. The enzyme catalyzes the removal of pyroglutamyl residues at the N-terminus of polypeptide chains. The polyamino acids used in these studies contained glutamic acid, aspartic acid, alanine, glycine, isoleucine, proline and valine. Alkaline hydrolysis was also used to determine indirectly that the N-termini of these polyamino acids are pyroglutamic acid. Another interesting finding was that many of the amino acids in the polymerization mixture were found to occur penultimate to the N-terminal amino acid. This is interpreted to mean that the diffusible fraction contains many polyamino acids.

Papillomas on fish exposed to chlorinated wastewater effluent.

The presence of carcinogenic and mutagenic chemical(s) in the effluent of a wastewater treatment plant was indicated by papillomas developing on caged black bullheads (Ictalurus melas), hepatic enzyme induction in exposed fish, and Ames test mutagenicity of organic extracts of the wastewater. Although virus-like particles have been reported in papillomas of several other fish species, no evidence was obtained for the presence of viruses in the black bullhead papillomas. Mutagenic and carcinogenic chemicals were not identified in the wastewater, but chlorination was implicated as a factor contributing to the induction of the papillomas. The prevalence of papillomas on wild black bullheads exposed to the effluent decreased from 73 to 23% after the amount of residual chlorine (CAS: 7782-50-5) in the effluent leaving the chlorine contact chamber was reduced from 1.3-3.1 mg/liter to 0.25-1.2 mg/liter.

Peptide separation and isolation with an automated amino acid analyzer.

A technique is presented for separation and collection of amino acids and peptides using a microcolumn amino acid analyzer. By use of the program and the column selection valve of the amino acid analyzer, and without any modification of the instrumentation, the stream of the eluate is diverted into the reaction coil and absorbance is recorded at regular intervals. The rest of the eluate is collected in a fraction collector for further characterization of the separated peptides. Splitting ratios can be varied by simple alteration of the program. Small amounts of material (1 to 2 nmol) are needed for monitoring the separation and collection of the eluate.

Comparative benzo[a]pyrene metabolite patterns in fish and rodents.

Benzo[a]pyrene is converted to 3-hydroxybenzo[a]pyrene, 9-hydroxybenzo[a]pyrene, benzo[a]pyrene-4,5-dihydrodiol, benzo[a]pyrene-7,8-dihydrodiol, benzo[a]pyrene-9,10-dihydrodiol, and benzo[a]pyrene quinones by postmitochondrial supernatant or microsomes in such fish as the rainbow trout, flounder, salmon, mullet, little skate, Fundulus grandis, and sea catfish. It is also now well-established that many fish convert benzo[a]pyrene to potent mutagenic metabolites as has been demonstrated with the Ames test, especially when the fish are induced with Aroclor 1254 or 3-methylcholanthrene. The metabolite patterns obtained at different substrate concentrations levels indicate that the metabolism is more complex at low concentrations when metabolites are recycled in the in vitro system.

Characterization of benzo[a]pyrene metabolites formed by 3-methylcholanthrene-induced goldfish, black bullhead and brown bullhead.

Metabolites of BaP formed by 3-MC-induced goldfish (Carassius auratus), black bullhead (Ictalurus melas), brown bullhead (Ictalurus nebulus) and rat have been separated by HPLC. The main metabolites in both bullhead species and the rat were 3-hydroxy-BaP and BaP 9,10- and 7,8-dihydrodiols, whereas BaP 1,6- and 3,6-diones and 9- and 3-hydroxy-BaP predominated in the goldfish. Induced rat formed over 10 times as many total metabolites as the fish. No induction was observed in the 3-MC-treated brown bullheads kept at 7 degrees C. TCPO and alpha-naphthoflavone decreased metabolite production by 50 and 30%, respectively.

Structure of thermal polymers of amino acids.

When glutamic acid is a predominant amino acid in a thermally polymerized mixture of amino acids, pyro Glu is exclusively found at the N-terminal end of the poly-amino acid polymer. It probably initiates the polymerization process. Lysine-containing polymers will probably contain epsilon N-(glutamyl)L-lysine cross links which may account for the higher molecular weights observed in these polymers (100-200 000). Incorporation of some amino acids facilitates the incorporation of others. When utilizing mixtures of three to eight amino acids with glutamic acid as one of the amino acids, some fractions are obtained which include all the amino acids in the polymerization mixture. The biosynthesis of glutathione, gramicidin, tyrocidine and cell-wall polypeptides has demonstrated that non-random amino acid sequence peptides can be biologically synthesized without the direct participation of nucleic acids. That is, the enzymes appear to provide adequate chemical specificity to form non-random amino acid sequence peptides. The properties and replication of the scrapie agent may provide us with more profound insight as to the evolution of purely physical-chemical systems into biological systems.

Effects of platinum complexes on chymotrypsin.

A variety of platinum complexes were evaluated as inhibitors of alpha-chymotrypsin. A standard enzyme assay was used with benzoyl-L-tyrosine ethyl ester as the substrate. Rb2PtBr4, trans-Pt(NH3)2Cl2, and K2PtCl4 were all effective enzyme inhibitors. Pt(Gly-L-Met(Cl2, Pt(Met)(NH3)2Cl, cis-Pt(NH3)2Cl2, and two ethylenediamminedichloroplatinum complexes were weak inhibitors of alpha-chymotrypsin. A surprising finding was the noninhibition of an immobilized preparation of alpha-chymotrypsin by the above inhibitors and by active-site-directed modifying reagents.

The biological activity of vitamin B6 analogs in the rat.

The effects of three vitamin B6 analogs were studied in young adult and weanling rats. The 2-ethyl analog was the most active. It produced higher growth rates, an average feed efficiency equal to the control and elevated liver glycogen levels. Xanthurenic acid excretion remained low with the 2-ethyl analog and pyridoxine in the diet and plasma amino acid concentration was low. Response to the 2-n-propyl analog was similar to that of a B6-deficient diet, with a large increase in xanthurenic acid excretion. Rats receiving the 2-isopropyl analog were intermediate between the ethyl group and B6-deficient animals in all parameters measured. These in vivo results parallel those reported in the in vitro yeast pyridoxine dehydrogenase enzyme system. The ethyl pyridoxine analog was the most active of all the analogs in the yeast dehydrogenase system, in supporting growth and in maintaining normal xanthurenic acid excretion in the rat.

Light-induced enzyme system leading to pigmentation.

Alpha-Chymotrypsin was light sensitized by acylating with cis-cinnamoyl ester, a substrate interconvertible to the trans form by ultraviolet (UV) light. The degree of acylation by this method was complete leaving no residual activity of the enzyme. Upon UV irradiation the inhibited enzyme regained about 70% of its original activity, thereby adding light-sensitiveness to the proteolytic enzyme. In seeking a photographic application of the light-sensitized enzyme, a pigmenting enzyme was incorporated with it. The coupled enzyme system was shown to exhibit a light signal in the form of dark pigment slurry.

An improved method of light-induced pigmentation.

An improved procedure was developed whereby a primary light signal can be intensified and made visible by activation of a pre-tyrosinase (pre-phenoloxidase) enzyme [isolated from silkworm (Bombyx mori)] by alpha-chymotrypsin; this activation results from the light-activated conversion of the inactive cis-cinnamoyl-alpha-chymotrypsin.

Enhanced inhibition of both cellular protein synthesis and malate dehydrogenase by aged aquoplatinum(II) complexes.

Previous studies have shown cis-diamminedichloroplatinum(II) (Cis) an effective anti-tumour agent in man and animals. Evidence is presented here that formation of aquo complexes of this platinum derivative will significantly enhance its inhibitory properties with respect to two separate biochemical functions, namely inhibition of protein synthesis in hamster medulloblastoma cells and in inhibiting the activity of L-malate dehydrogenase (MDH) in a cell free system. Inhibition of cell protein synthesis rises from 8% using freshly dissolved drug to 30% when aged solutions of drug are employed at an inhibitor concentration of 0.1 mM. The inhibitory enhancement seen using purified malic dehydrogenase increases from 16% (fresh) to 57% (aged) at an inhibitor concentration of 1 mM.

Interactions of cis- and trans-platinum(II) complexes with dehydrogenase enzymes in the presence of different mono- and polynucleotides: evidence for a ternary complex.

The inhibition of several dehydrogenase enzymes by cis- and trans-Pt(NH3)2Cl2 have been measured in the presence of baker yeast ribonucleic acid (RNA), calf thymus and salmon sperm deoxyribonuclic acid (DNA) and several mononucleotides (AMP and ATP). The binding constants for the interaction of the platinum complexes to the nucleotides have been calculated and a comparison of those values to the previously calculated platinum complex-enzyme binding constants strongly suggest that platinum compounds are more tightly bound to the enzymes. The binding of the platinum complexes to most of the enzymes was decreased in the presence of any nucleotide, yet it was observed that when using rabbit muscle (M4) lactate dehydrogenase the mononucleotides reduced the binding to a lesser degree while the polynucleotides actually enhanced the platinum-enzyme interaction. The implications of these interactions are discussed.

A proposed mechanism relating the antitumor behavior of cis-platinum amine complexes to their inhibition of a model enzyme.

The twenty-four hour inhibition of m-malate dehydrogenase (E.C. 1.1.1.37) by various complexes of cis-platinum(II) and cis-platinum(IV) was measured as a function of the platinum concentration. It was observed that increased alkylation of the amine groups of Pt(II) and to a lesser degree of Pt(IV) decreased the activity consistently. It was also observed that the Pt(IV) analogues inhibit the enzyme to about an order of magnitude greater than the Pt(II) complexes. These phenomena will be interpreted.

Effects of divalent cations and sodium taurocholate on pancreatic lipase activity with gum arabic-emulsified tributyrylglycerol substrates.

The effects of Ca2+ and/or sodium taurocholate on lipase activity with gum arabic-emulsified tributyrylglycerol substrates were investigated. Calcium was found to slightly increase lipase activity while bile salts showed marked inhibition except at very low concentrations. Calcium eliminated inhibition seen with low concentrations of bile salts and reduced the inhibition seen at higher bile shift of the enzyme from the alkaline region in the absence of bile salt to the slightly acidic region in the presence of bile salt. Calcium was shown to eliminate the time lag periods between enzyme addition and maximum rate of hydrolysis seen at low substrate concentrations and the time lag noted when bile salts were included with normal (substrate concentration not limiting) assay concentrations of substrate. Zeta potential measurements indicated that Ca2+ reduced the negative charge on the gum arabic-emulsified particle while bile salts did not increase the negative charge. Commercial preparations of gum arabic were found to have significant concentrations of Ca2+ and Mg2+.

Interactions of platinum complexes, peptides, methionine and dehydrogenases.

Eight platinum-methionine complexes have been investigated as inhibitors of the alcohol (EC 1.1.1.1) and lactate (EC 1.1.1.27) dehydrogenase systems. While only one complex, Pt(Met)Cl2, had two halogen ligands on the platinum, all the complexes were able to inhibit both enzyme systems. Of the various methionine and histidine containing peptides evaluated, the dipeptide Met-Met gave the best protection against inhibition by N-alkyl-substituted ethylenediamine-platinum complexes. The histidine containing peptides gave a slight protection against the inhibition by beta-[Pt(Met)(NH3)Cl]Cl. Thus it appears that in the enzyme systems studied, the methionine acts as a strong ligand for platinum.