Katsunobu Takahashi

Application of polyethylene glycol-modified enzymes in biotechnological processes: organic solvent-soluble enzymes

Abstract A new approach in biotechnological processes is to use enzymes modified with polyethylene glycol which has both hydrophilic and hydrophobic properties. The modified enzymes are soluble in organic solvents such as benzene, toluene and chlorinated hydrocarbons and exhibit high enzymic activities in these organic solvents. Modified hydrolytic enzymes catalysed the reverse reaction of hydrolysis in organic solvents: formation of acid—amide bonds by modified chymotrypsin, and ester synthesis and ester exchange reactions by modified lipase. Modified catalase and modified peroxidase efficiently catalyse their respective reactions in organic solvents. The results of this research indicate great potential for applications in the fields of biotechnology and enzymology.

Ester synthesis catalyzed by polyethylene glycol-modified lipase in benzene

Lipoprotein lipase, which catalyzes hydrolysis of emulsified triglycerides or water-insoluble esters, was modified with 2,4-bis(o-methoxy-polyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified lipase, in which 55% of the total amino groups in the lipase molecule, was soluble in organic solvents such as benzene, toluene, chloroform and dioxane. The modified lipase could catalyze ester synthesis reaction in benzene. When very hydrophobic substrates of lauryl alcohol and stearic acid were used, the ester synthesis reaction proceeded efficiently in the transparent benzene solution with the maximum activity of approximate 5.0 mumoles/min/mg of protein. Ester exchange and aminolysis reactions were also conducted with the modified lipase in benzene.

A chemical modification to make horseradish peroxidase soluble and active in benzene

Horseradish peroxidase was modified with 2,4-bis(O- methoxypolyethylene glycol)-6-chloro-s-triazine. The modified peroxidase, in which 60% of the amino groups were coupled with polyethylene glycol, had 70% of the enzymic activity in aqueous solution and was found to be soluble in benzene. Since the modified peroxidase in benzene had an absorption spectrum similar to that of unmodified peroxidase in aqueous solution, the prosthetic group, protohaemin IX, remained with the apoprotein even in benzene. The modified peroxidase in benzene had 21% of the enzymic activity relative to that of unmodified enzyme in aqueous solution.

Endothelin induces two types of contractions of rat uterus: Phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using 125I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.

Polyethylene glycol-modified enzymes trap water on their surface and exert enzymic activity in organic solvents

SummaryPolyethylene glycol-modified enzymes dissolved and had high enzymic activity in organic solvents. A trace amount of water was found to be necessary for the activity. It was reasoned that the amphipathic polymer covalently attached to enzymes kept water molecules around them. This was supported by findings that : (1) high enzymic activity was found in water- immiscible solvents, whereas activity was never observed in water-miscible solvents; (2) enzymic activity was inhibited by increasing the concentration of dimethyl sulfoxide in benzene; (3) activity of lipase was inhibited by a water-miscible alcohol substrate, but was steadily elevated by increasing the concentration of a water-immiscible alcohol substrate; (4) water was not absorbed from benzene solution containing a modified enzyme by molecular sieves, while it was easily absorbed in the presence of a water-miscible organic solvent, dimethyl sulfoxide.

Mapping of G Protein Coupling Sites of the Angiotensin II Type 1 Receptor

Angiotensin II type 1 (AT1) receptors have been identified in a wide variety of tissues, including the kidney, liver, adrenal gland, cardiovascular system, and brain. AT1 receptors also mediate complex signaling mechanisms that elicit a diversity of specific physiological effects. The rat AT1A receptor has seven transmembrane domains and couples with three distinct G proteins: Gq, Gi, and Go. But it is unknown which domains of AT1A couple with and activate each type of G protein. To identify the domains responsible for the activation of various types of G protein, we studied the effect of five different synthetic peptides representing different domains of cytosolic segments of the rat AT1A receptor on the binding of the 35S-labeled stable analogue of GTP, GTP gamma S. Peptides P-3, which is located in the N-terminal region of the putative third intracellular loop of AT1A (residues 216 through 230), and P-5 (residues 306 through 320), corresponding to the N-terminal region of the C-terminal tail, were found to activate purified Gi1, Gi2, and Go proteins. These results indicate that not only the third cytosolic loop but also the C-terminal cytosolic domain of AT1A is important for Gi1, Gi2, and Go protein coupling and activation.

ACCUMULATION OF CYCLIC ADP-RIBOSE MEASURED BY A SPECIFIC RADIOIMMUNOASSAY IN DIFFERENTIATED HUMAN LEUKEMIC HL-60 CELLS WITH ALL-TRANS-RETINOIC ACID

Cyclic adenosine diphosphoribose (cADPR) is a novel candidate for the mediator of Ca2+ release from intracellular Ca2+ stores. The formation of this cyclic nucleotide is catalyzed by not only Aplysia ADP‐ribosyl cyclase but also an ecto‐form enzyme of NAD+ glycohydrolase (NADase), which was previously identified as all‐trans‐retinoic acid (RA)‐inducible CD38 in human leukemic HL‐60 cells. In the present study, we developed a radioimmunoassay specific for cADPR, by which more than 100 fmol of cADPR could be detected without any interference by other nucleotides. The possible involvement of CD38 in the formation of cellular cADPR was investigated with the radioimmunoassay method. A marked increase in cellular cADPR was accompanied by all‐trans‐RA‐induced differentiation of HL‐60 cells. Moreover, a high level of cellular cADPR was observed in other leukemic cell lines, in which CD38 mRNA was expressed. Thus, CD38, which was initially identified as an NADase, appeared to be responsible for the formation of cellular cADPR.

Polymerization of 10-hydroxydecanoic acid in benzene with polyethylene glycol-modified lipase

SummaryA hydrophobic substrate, 10-hydroxydecanoic acid having two functional groups (−OH and −COOH) in the molecule, was polymerized by ester bond formation with the polyethylene glycol-modified lipase in a transparent benzene solution. The polymer of 10-hydroxydecanoic acid was linearly elongated under a quite mild condition.

Polyethylene glycol-modified catalase exhibits unexpectedly high activity in benzene

Bovine liver catalase with molecular weight of 248,000, which consists of four subunits, was modified with 2,4-bis(o-methoxypolyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified catalase became soluble in organic solvents such as benzene by increasing the degree of modification of amino groups in the enzyme with activated PEG2. The enzymic activity of the modified catalase in benzene, in which 42% of the total amino groups were coupled with the modifier, was unexpectedly high in comparison with the activity of non-modified catalase in aqueous system. The absorption spectrum of the modified catalase in benzene showed the characteristic pattern of a haem protein with Soret band at 405 nm. The temperature-activity profile of the modified catalase in benzene was clarified and its activation energy was estimated to be 1900 cal/mol.

In Vivo Evidence for the Link Between l‐ and d‐Serine Metabolism in Rat Cerebral Cortex

Abstract: To obtain an insight into the metabolic pathways of endogenous d‐serine in mammalian brains, we have investigated in the infant rat the effects of systemic administration of l‐serine, d‐serine, and related amino acids, including glycine and threonine, on the amino acid contents in the cerebral cortex. Intraperitoneal injection of l‐serine induced a rapid and transient elevation of the levels of l‐serine itself in the neocortex, with its peak at 3 h post injection, and a delayed and prolonged increase in d‐serine contents from 1.5 h to at least 24 h thereafter. Similarly, a significant augmentation in cerebral d‐serine contents was observed 6 h after intraperitoneal administration of glycine, which also elevated the cortical l‐serine levels. In contrast, l‐threonine injection affected the concentrations of neither d‐ nor l‐serine in the cortex of the pups. d‐Serine given systemically, in turn, increased the neocortical contents of l‐serine as well as d‐serine itself, but failed to alter those of glycine and l‐threonine. These in vivo data suggest the possible link between metabolic pathways of d‐ and l‐serine in the cerebral cortex of the rat.