A biochemical logic gate using an enzyme and its inhibitor. 1. The inhibitor as switching element

Molecular-scale logic systems will allow for further miniaturization of information processing assemblies and contribute to a better understanding of brain function. Of much interest are the pertinent biological systems, some of the basic components of which are biomolecular switching elements and enzyme-based logic gates.In this series of accounts, results of investigations are presented on the implementation of an enzyme/inhibitor logic gate operating under the rules of Boolean algebra. In this report (part 1 of the series), consideration is given to the experimental conditions-particularly the irradiation mode-that affect the performance of proflavine as inhibitor of alpha-chymotrypsin. Also, assessments are made on the reversibility of the process involved and the long-term stability of the system. Moreover, using a theoretical conformational analysis of proflavine and its reduction products, detailed features were established regarding their three-dimensional structure, partial charge distribution, and hydrophobicity. Accordingly, an understanding was reached as to the factors affecting the interaction between these compounds and the enzyme. In part 2 of this series, the actual implementation of an AND logic gate will be presented. This gate involves proflavine and a chemically derivatized alpha-chymotrypsin, and its operation relies on the conclusions reached in this report regarding the optimal mode for controlling the inhibitory activity of proflavine.     

A reversible DNA-silver nanoclusters-based molecular fluorescence switch and its use for logic gate operation

Molecule-like silver nanoclusters (AgNCs) with few to tens of atoms are highly sensitive to the sequence and structure of DNA stabilizers. In this paper, a novel pH-triggered reversible molecular fluorescence switch is developed by taking advantage of the DNA-dependent fluorescence pH response of AgNCs. The DNA-AgNCs fluorescence switch simultaneously addresses concerns of simple construction strategy, efficient design and organic-solvent-free operation. Moreover, the excellent photostability and biocompatibility of AgNCs provide great potential for application of the DNA-AgNCs fluorescence switch in the development of functional molecular devices. Specifically, we apply the DNA-AgNCs fluorescence switch combined with the DNA sequence-dependent pH response pattern of AgNCs for construction of molecular logic gates.     

2,4-Diaminopyrimidine MK2 inhibitors. Part II: Structure-based inhibitor optimization

We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC₅₀ values as low as 19 nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFα production in peripheral human monocytes.     

Essential hypertension: effect of an oral inhibitor of angiotensin-converting enzyme.

Captopril, a specific oral inhibitor of angiotensin-converting enzyme, was given to 18 unselected patients with moderate essential hypertension. Mean blood pressure fell by 14.5% at the maximum dose given, and this fall was significantly correlated with the initial plasma renin activity. The main fall in blood pressure occurred two hours after the first dose of captopril. These results suggest that captopril effectively lowers blood pressure in patients with essential hypertension and that the renin-angiotensin aldosterone system may maintain blood pressure in essential hypertension. This does not necessarily imply that the renin-angiotensin system is the cause of the high blood pressure.     

Studies on phospholipase A inhibitor in blood plasma. II. Interaction of phospholipase A inhibitor with phospholipase A and its specificity

Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.     

The hemicelluloses of bracken Part II. A galactoglucomannan

A galactoglucomannan has been isolated from the stem tissues of a fern, bracken (Pteridium aquilinum). It had [] −35° and, on hydrolysis, yielded mannose, glucose, and galactose in the molar ratios of 60:15:1. Mild hydrolysis with acid released galactose only. The d.p., determined by periodate-oxidation and Smith-degradation studies, was 43–45 for a doubly branched molecule. The methylated galactoglucomannan had a d.p. of 25–30 by ebulliometry. Methylation analysis, in combination with other evidence, indicated that the hemicellulose had β-(1→4)-linked -glucopyranose and -mannopyranose residues in the ratio of 1 to 4, with residues of the latter contiguous. -Mannopyranosidic and -galactopyranosidic residues were present as non-reducing end-groups in the ratio of 7 to 1, but there was no evidence of non-reducing -glucopyranosidic residues. The branching was to 6-positions on the main glucomannan chain and was probably -(1→6). The galactoglucomannan is similar to hemicelluloses isolated from softwoods, but the former has a lower d.p.     

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part II: Single-step procedure using a coimmobilized enzyme system

The enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid (7-ACA) using coimmobilized -aminoacid oxidase (DAAO) and 7-β-(4-carboxybutanamido)cephalosporanic acid acylase (Gl-7-ACA acylase) is reported. The results from the coimmobilization of the two enzymes on different carriers and at different ratios of enzyme activities are described. When an inhibitor of catalase activity, such as NaN₃ or H₂O₂, is present, the conversion rate to 7-ACA is higher, but more by-products are obtained. An optimum ratio of 60:1 between the enzymatic activities of DAAO and Gl-7-ACA acylase in the coimmobilized sample at 0.21 Ug⁻¹ Gl-7-ACA acylase activity was determined. The results of using coimmobilized enzymes and of using a mixture of separately immobilized enzymes in the same process are compared.     

A reappraisal of the terverticillate penicillia using biochemical, physiological and morphological features. II. Identification

The data from an integrated numerical classification was used to construct identification schemes for some fasciculate penicillia. The identification schemes were presented as a synoptic key and a frequency matrix for computer-assisted identification. Statistical testing of the frequency matrix showed that although character separation values were generally low, only four pairs of taxa showed overlap greater than that expected for a rectangular distribution. The identification schemes were tested practically with 52 previously studied strains and 51 further cultures. A synoptic key based on 10 and 90% cutoff limits was used to correctly identify 44 of the 51 additional strains, although this proved very sensitive to single test discrepancies. The frequency matrix was used to correctly identify 45 of the additional strains with a Willcox probability score and this was compared to identifications based on the modal likelihood fraction.     

A novel structure of an antikinase and its inhibitor

In Bacillus subtilis, the KipI protein is a regulator of the phosphorelay governing the onset of sporulation. KipI binds the relevant sensor histidine kinase, KinA, and inhibits the autophosphorylation reaction. Gene homologues of kipI are found almost ubiquitously throughout the bacterial kingdom and are usually located adjacent to, and often fused with, kipA gene homologues. In B. subtilis, the KipA protein inhibits the antikinase activity of KipI thereby permitting sporulation. We have used a combination of biophysical techniques in order to understand the domain structure and shape of the KipI-KipA complex and probe the nature of the interaction. We also have solved the crystal structure of TTHA0988, a Thermus thermophilus protein of unknown function that is homologous to a KipI-KipA fusion. This structure, which is the first to be described for this class of proteins, provides unique insight into the nature of the KipI-KipA complex. The structure confirms that KipI and KipA are proteins with two domains, and the C-terminal domains belong to the cyclophilin family. These cyclophilin domains are positioned in the complex such that their conserved surfaces face each other to form a large “bicyclophilin” cleft. We discuss the sequence conservation and possible roles across species of this near-ubiquitous protein family, which is poorly understood in terms of function.     

The intraglobular electrostatic field of an enzyme. II. Effect of environment polarization

The influence of polarization of surrounding medium on the intraglobular electric field of the alpha-chymotrypsin molecule is considered. The polarization is taken into account by the image charges method, the proper approximations for calculation of the fields due to intraglobular and surface charges are suggested. The polarization of surroundings does not change the qualitative picture of the electric field in the active center of the alpha-chymotrypsin molecule set up by protein dipoles, but reduces almost to zero the intraglobular field set up by surface ions.     

Guanyl cyclase in Escherichia coli. II. Identification and characteristics on the enzyme inhibitor]

The activity of guanylate cyclase and that of its inhibitor present in E. coli extract, have been separated through a linear KCl gradient on DEAE-cellulose column. The activity of the inhibitor is lost after ribonuclease treatment, whereas is strengthened by addition of poly (C). Other types of RNA synthetic homopolymers do not affect the inhibitor's activity. Chromatographic analysis of the products of guanylate cyclase measured in the presence of FI and FI plus poly (C), indicated that the inhibitor has a poly (C) dependent GTPase activity.