Synthesis of polypeptides by microwave heating II. Function of polypeptides synthesized during repeated hydration-dehydration cycles

Summary Polypeptides, synthesized from a mixture of amino acid amides by microwave heating during repeated hydration-dehydration cycles, showed hydrolase- and oxidoreductase-like catalytic activities. Poly(GAVDH), polypeptides synthesized from an equimolar mixture (each 0.1 M) of glycinamide,l-alaninamide,l-valinamide,l-aspartic acid -amide, andl-histidinamide, catalyzed the hydrolysis of PNPAc. The hydrolytic rate of PNPAc with poly(GAVDH) was the quadruple of that ofl-histidine alone. Though the k_cat values of different resulting polypeptides were 10³–10⁶ times less than those of native hydrolases, the K_m value of the polypeptides further containing phenylalanine residues was nearly equal to that of the esterase. This result indicates the presence of hydrophobic interaction between a substrate and the polypeptides. Resulting polypeptides also showed catalytic activity for the reduction of ferricyanide ion [Fe(CN)^3–] with NADH. The polypeptides seemed to have a strong affinity for adenine nucleotides, because the reaction was inhibited by adenine derivatives such as NAD⁺ and AppA. A hypothesis for the emergence of primitive protein enzymes is discussed.     

Synthesis of polypeptides by microwave heating I. Formation of polypeptides during repeated hydration-dehydration cycles and their characterization

Summary Amino acid amides effectively reacted to produce polypeptides in response to microwave heating during repeated hydration-dehydration cycles. The polypeptides, formed from a mixture of glycinamide, alaninamide, valinamide, and aspartic acid -amide, had molecular weights ranging from 1000 to 4000 daltons. Amino acids were incorporated into the polypeptides in proportion to the starting concentrations, with the exception of glycine whose incorporation was 1.5 times higher than that of the other amino acids. The polypeptides had some definite secondary structure, such as -helix and -sheet, in aqueous solution. This reaction provides not only a convenient method for abiotic peptide formation but also a convenient method for the chemical synthesis of peptides.     

Endotoxic properties of chemically synthesized lipid A analogs. Studies on six inflammatory reactions in vivo, and one reaction in vitro

Biological activities of two groups of synthesized lipid A analogs, the counterpart of biosynthetic precursor, Lehmann's Ia type, 406, and E. coli lipid A type, 506, as well as their non-phosphorylated, and mono-phosphorylated analogs were investigated. The activities employed included four bone marrow cell reactions in mice, mice skin reaction, leukocytes migration in rabbits' cornea, and hemagglutination. Compound 406 and 506 elicited bone marrow reactions in mice and hemagglutination of mouse RBC, although 406 failed to elicit hemorrhage and necrosis also in mice skin. Compound 406 did not elicit corneal reaction in rabbits. The results suggest that for elicitation of this reaction and mice skin reaction, acyloxyacyl structure is required. Cytotoxicity and thromboplastin production of four bone marrow reactions had been reported by us to be endotoxic reactions, since these had not been elicited by peptidoglycan of Lactobacillus and Staphylococcus (1981) and 300 series synthesized analogs (1984) which did not have endotoxic structures. From these results, it seems that these two marrow reactions and hemagglutination require, as does the limulus test, the lipid A part structure as is present in 406.     

Synthetic inositol trisphosphate analogs and their effects on phosphatase, kinase, and the release of Ca2+

A series of inositol 1,4,5-trisphosphate (IP3) analogs and positional isomers was examined to explore the structure-activity relationships among IP3 5-phosphatase, IP3 3-kinase, and the release of Ca2+. All analogs with additional groups on the 2nd position of IP3 inhibited the hydrolysis of [5-32P]IP3 catalyzed by erythrocyte ghosts, with a lower Ki value than seen with IP3. IP3 dehydroxylated at the 2nd position also had a lower Ki, while 2,4,5-IP3 or cyclic(1:2), 4,5-IP3 had higher Ki values. Among these compounds 2-deoxy-IP3 was as potent as IP3 in inhibiting the phosphorylation by [3H] IP3-3-kinase in rat brain cytosol. The other compounds, except for 2,4,5-IP3 inhibited the phosphorylation, however, 2-30 times higher concentrations were required. By lowering free Ca2+, the concentrations required for half-maximal inhibition were low, while those of IP3, 2-deoxy-IP3, and positional isomers remained unchanged. These compounds acted as full agonists in releasing Ca2+ from permeabilized macrophages, although 1.6-50-fold higher concentrations than IP3 were required. These compounds also inhibited the binding of [3H]IP3 to rat cerebellum and bovine adrenal cortex microsomes, but the potencies were 2.9-33 times less than that of IP3. Thus, the 2nd position of IP3 can be modified with only a slight loss of biological activity.     

Intramolecularly quenched fluorogenic peptide substrates for human renin.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.     

Pyrogenic action of endothelin in conscious rabbit.

Injection of 0.3 nmol/kg endothelin(ET)-1 into the ear vein of conscious rabbits induced a significant increase in body temperature. ETB receptor specific agonist, namely 4-Ala-ET-1, also caused an elevation of the body temperature in a dose-dependent manner by injection into the ear vein of rabbit. These results suggest that ET play important roles in regulation of body temperature through selective stimulation of ETB receptor.     

Endothelin receptors in human parathyroid gland.

We studied whether specific receptors for endothelins (ETs) exist in human parathyroid tissues and whether ETs may have any effect on secretion of PTH from parathyroid cells. Binding studies using [125I]ET-1 to the parathyroid membranes obtained from patients with hyperparathyroidism (2 adenomas, 2 hyperplasias) revealed that ET-1 competitively inhibited the binding of [125I]ET-1 to the membranes (the apparent Kd: 62 +/- 18 pM), whereas ET-3 showed biphasic and less steep inhibition curve than ET-1 in all tissue membranes examined. Northern blot analysis of poly(A)+ RNA from the parathyroid adenoma clearly demonstrated gene expression of both ETA and ETB receptors as well as preproET-1. ET-1 inhibited basal PTH secretion from dispersed adenoma cells more potently than ET-3. The present study clearly demonstrates the presence of both ETA and ETB receptor subtypes in human parathyroid tissues through which ETs may modulate PTH secretion in an autocrine and/or paracrine manner.     

Characterization of atrial natriuretic peptide in urine from rats treated with a neutral endopeptidase inhibitor.

To explore the mechanisms for the natriuretic effects of a neutral endopeptidase inhibitor, candoxatril, the concentration of atrial natriuretic peptide (ANP) and its molecular forms in the urine of Dahl salt-sensitive (S) rats were examined. Candoxatril-induced natriuresis (+120%, p less than 0.05) was associated with a marked increase in the urinary ANP excretion (+1200%, p less than 0.05). Analysis by Sephadex G-50 gel filtration revealed that molecular weight of the major fraction of immunoreactive (ir-) ANP in the plasma of candoxatril-treated Dahl S rats was 3K, whereas that in the urine was 2.5 K. Further analysis by reverse phase high performance liquid chromatography showed that ir-ANP in the plasma of Dahl S rats was alpha-rANP (1-28), while that in the urine from rats treated with candoxatril was alpha-rANP (1-25). These results indicate that candoxatril inhibits the complete degradation of ANP in the kidney, thereby increasing the amount of biologically active ANP reaching the distal nephron and contributing to natriuresis.     

Initiation of DNA synthesis in the transfer origin region of RK2 by the plasmid-encoded primase: detection using defective M13 phage

The broad host range IncP (IncP1) plasmids of gram-negative bacteria encode DNA primases that are involved in conjugal DNA synthesis. The primase of RK2/RP4 is required for efficient DNA transfer to certain gram-negative bacteria, indicating that the enzyme primes complementary strand synthesis in the recipient. In vitro, the primase initiates synthesis of oligoribonucleotides at 3'-dGdT-5' dinucleotides on the template strand. In this report, replication-defective M13 phage are used to assay the ability of the RK2-encoded primase to initiate complementary strand synthesis in vivo on single-strand templates containing the RK2 origin of conjugal transfer (oriT) or the RK2 origin of vegetative replication (oriV). The results show that sequences from either strand of the oriT region serve as efficient substrates for the RK2 primase and can enhance the growth of the defective M13 vectors delta E101 and delta Elac to levels approaching wild-type. The primise-oriT interaction appeared specific, since neither the oriV sequence nor another RK2 region, trfB, significantly enhanced growth of the defective phage, either in the presence or in the absence of the primase. In contrast to ColEl and F, this study also shows that the oriV region of RK2 lacks sites that are recognized by the host-specified DNA priming systems. The results suggest that the oriT region contains sites on both DNA strands that are efficient substrates for the plasmid-encoded primase, facilitating initiation of complementary strand DNA synthesis in both donor and recipient during conjugation.     

Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.