Search

W39F, F52Y, S98G, S98A, and S98C mutants of the neocarzinostatin apoprotein (apo-NCS) were newly prepared and investigated their physicochemical properties. The circular dichroism (CD) spectra of F78W, F52Y, S98A, S98G, S98C were superimposable with that of wild type 1R49 protein although the minor spectral change seemed to be in the ellipticity of W39F. The results suggest that position 52, 78, and 98 involving natural chromophore binding do not play a major role in the inducing overall structural changes of the protein. Conversely, the position 39 would be affected slightly. Ethidium bromide (EtdBr) binding to mutants was also evaluated by the monitoring of total fluorescence intensity and fluorescence polarization (FP). The observed dissociation constant in the FP study was 4.4 microM for wild type, 2.2 microM for S98A, 1.3 microM for S98G, 9.7 microM for S98C, respectively. When S98G and F52Y, the calculated maximum change of the total fluorescence intensity was increased, suggesting that the EtdBr binding to S98G or F52Y were slightly improved compared with the wild type. Then, a total of 14 amino acids randomly substituted phage displayed library of apo-NCS was successfully prepared, because substitution of the amino acid structured the chromophore-binding cavity were not change the overall structural features. The phages which bound glycyrrhetic acid conjugated bovine serum albumin were enriched from this library using phage display technique as the pilot experiments. Although more precision investigation still needs, it should be possible to select variants that have new functions not found in nature.

We studied the steric course of the reaction catalyzed by the N-ethylmaleimide (NEM) reductase of Yarrowia (Candida) lipolytica (Y. lipolytica), using 4R-[4-2H1]NADPH and 4S-[4-2H1]NADPH as cofactors and N-ethylcitraconimide as substrate. Active substrates and inhibitors of NEM reductase and its subcellular distribution were also investigated to clarify the biochemical properties of this enzyme. NEM reductase catalyzes the reduction of N-ethylmaleimide to N-ethylsuccinimide with NAD(P)H as the cofactor. Several maleimide and cyclopentenone derivatives tested were also active substrates for NEM reductase of Y. lipolytica. Some pyrazolone derivatives, particularly 1-phenyl-5-pyrazolone, were found to be effective inhibitors of NEM reductase. Subcellular localization of NEM reductase was carried out using protoplast formation and differential centrifugation. Ninety-eight percent of the NEM reductase activity was recovered in the cytosolic fraction, indicating that NEM reductase in Y. lipolytica was the cytosolic enzyme. We also determined the stereochemical specificity of the reduction of N-ethylcitraconimide by NEM reductase in Y. lipolytica, showing that 4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by NEM reductase, and two hydrogen atoms from NADPH and H2O added to opposite faces of the double bond of N-ethylcitraconimide.

A monoclonal antibody against NCS-chr was prepared and characterized. Because of the instability of NCS-chr, chemically synthesized stable analog compound, termed PS, was used as a hapten of immunogen. The obtained antibody, termed APS, reacted with NCS-chr, but neither with NCS, NCS-polystyrene-maleic acid conjugate (SMANCS), nor UV-irradiated NCS-chr. Epitope analysis using the compounds that have a structure similar to PS showed that APS recognized the total structure, particularly cyclopenten moiety, of PS. These results suggest that APS recognizes the enediyne structure of NCS-chr. Next, the inhibition enzyme-linked immunosorbent assay (ELISA) for determination of NCS-chr was established. The standard curve showed that the microgram order of NCS-chr were accurately measurable by the established ELISA. Furthermore, it was revealed that the established ELISA was more sensitive than the antibiotic activity determination, termed Bio-assay. The established ELISA will be useful as a quantitative method of NCS-chr.

Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from tri-O-acetyl-D-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of 3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs.

NMR studies indicatethe hydrophobic pocket formedby the internalfour-stand β-sheet of apoprotein bindsNCS-chr with the conformation in wich its naphthoate moiety sits on the bottom of the pocket and the aminosugar and the carbonate group face outwards.

Neocarzinostatin (NCS), an antitumor protein antibiotic, is composed of apo-neocarzinostatin (apo-NCS) and neocarzinostatin-chromophore (NCS-chr), the principle of the biological activities of NCS. Apo-NCS having two tryptophan (Trp) residues at positions (39 and 83) was chemically modified by N-bromosuccinimide in a study on the correlation of the binding site(s) of NCS-chr. Selective oxidation of Trp residues was observed when NCS was titrated with N-bromosuccinimide. In contrast, non-selective oxidation of the two Trps on apo-NCS was observed and both Trp (39 and 83) of apo-NCS were titrated with N-bromosuccinimide. After selective oxidation, the remaining Trp residue of NCS was assigned as Trp (83). These results clearly indicate that the Trp (83) residue of apo-NCS changed from the "reactive type" to the "non-reactive type" after the binding of NCS-chr with apo-NCS. The fluorescence emission intensity of apo-NCS generated from the Trp (39) residue was quenched by NCS-chr. These data suggest that NCS-chr directly interacts with the Trp (39) residue and that a beta-sheeted loop containing the Trp (83) residue of apo-NCS changes the high-order structure upon binding with NCS-chr.

The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3,trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxyacyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240,000. Two types of subunits, with molecular weights of 73,000 and 42,000, were identified. The complex was composed of two copies each of the 73,000- and 42,000-Da subunits. The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.

Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H)-quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6-[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (K(m) = 5.26 muM, intrinsic clearance (CL(int)) = 0.34 microl/pmol P450/min), CYP1B1 (K(m) = 11.2 microM, CL(int) = 0.03 microl/pmol P450/min), and CYP3A5 (K(m) = 2.89 microM, CL(int) = 0.05 microl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (K(m) = 1.60 microM, CL(int) = 0.57 microl/pmol P450/min), CYP2C19 (K(m) = 5.95 microM, CL(int) = 0.16 microl/pmol P450/min), CYP3A4 (K(m) = 5.35 microM, CL(int) = 0.10 microl/pmol P450/min), and CYP2C8 (K(m) = 33.8 microM, CL(int) = 0.009 microl/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively.

Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from tri-O-acetyl-D-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of 3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs.

The purpose of this study was to evaluate the usefulness of extra-weak chemiluminescence (CL) measurement as a rapid method to estimate the stability of Kampo extract preparations. It was found that the Kampo drugs that emit little CL were stable, while those with higher CL were comparatively unstable with regard to the various stability markers, including change of coloration (browning), contents of specific ingredients, high molecular compounds, amino acids and sugars under various conditions of heat storage. Excellent correlation existed between the CL of Kampo drugs and the coloration (delta E* (ab)) and the other above-mentioned evaluation markers. From this investigation, it was deduced that the CL of Kampo drugs originates in the early stage of the Maillard reaction and reflects the stability of the preparations, and that CL is useful for estimating the stability of Kampo drugs.

[N-11C-methyl]-cocaine ([11C]cocaine), synthesized by N-methylation of norcocaine with [11C]CH3I, was used to assist in imaging the variety of local distribution by positron emission tomography (PET). The radiochemical yield and the radiochemical purity after purification of [11C]cocaine by high performance liquid chromatography (HPLC) at a sp. act. of 814GBq/mmol were 47–58% and > 99%, respectively. The time required for synthesis including the purification was 25–30 min from the end of [11C]CH3I trapping. The physical distribution of [11C]cocaine in organ was also investigated in mice at various time after i.v. injection. The main accumulation of radioactivity occurred in the lung, kidney and brain within l min after the injection. In the brain, no differences in the organ were observed except the radioactivity level in each section increased for the first 5 min, since then radioactivity decreased dramatically. Furthermore, in the behavioral sensitization model of cocaine, the peak of [11C]cocaine uptake in each brain area was shown to be 5–15 min.

Characteristics of the secondary structure of neocarzinostatin apoprotein (apo-NCS) were examined by various means. Gaussian analysis of the Fourier-transform infrared (FT-IR) curve and curve-fitting of the circular dichroism (CD) spectrum for apo-NCS revealed that this peptide was abundant in beta-structures. In the presence of sodium dodecyl sulfate (SDS), the CD bands of NCS originating from phenylalanyl, tyrosyl, and cystinyl residues decreased, indicating a conformational change around the chromophore (CNS-chr). On the other hand, apo-NCS, in the SDS system, showed no change of these bands. We showed that the major parts of the protein moiety consist of beta-structures by measurements of the FT-IR and CD spectra of apo-NCS and a prediction of the secondary structure based on the amino acid sequence of the peptide. It seems that properties of the protein may be important to the hydrophobic interaction between NCS-chr and apo-NCS.

The potassium permanganate oxidation of 3-methylthio-5, 6-diphenyl-as-triazine in the presence of a phase-transfer catalyst afforded 3-methylsulfonyl-5, 6-diphenyl-as-triazine. The 3-sulfonyl-as-triazine readily reacted not only with active methylene compounds but also with methyl or methylene ketones under basic conditions, and 5, 6-diphenyl-as-triazines containing a functionalized carbon substituent at the 3-position were obtained. Similarly, 2-substituted 4, 6-dimethylpyrimidines were synthesized by the nucleophilic substitution of 4, 6-dimethyl-2-phenylsulfonylpyrimidine with various ketones.

The structure of the chromophore of an antitumor antibiotic neocarzinostatin has been elucidated as a bicycro[7,3,0]dodecadiyne system having naphthalenecarboxylic acid, aminosugar and ethylene carbonate units.

The 1, 3-dipolar cycloaddition of 4-benzyloxy-6-methylpyrimidine 1-oxide (Ia) with dimethyl acetylenedicarboxylate (DMAD) gave dimethyl α-oxo-α'-(4-benzyloxy-6-methyl-2-pyrimidinyl) succinate (IIa), which was readily hydrolyzed to methyl 4-benzyloxy-6-methyl-2-pyrimidineacetate (IIIa). A similar reaction of 4-methoxy-(Ib) and 4-ethoxy-6-methylpyrimidine 1-oxide (Ic) gave corresponding methyl 2-pyrimidineacetates (IIIb, c). On the other hand, 6-methyl-4-piperidinopyrimidine 1-oxide (Id) reacted with DMAD to give a betaine derivative, 1-(6-methyl-4-piperidino-1-pyrimidinio)-1, 2-bis (methoxycarbonyl) ethenyl-2-oxide (IV).

A series of 5-amino-3-phenylisoxazole derivatives was synthesized and evaluated as part of a search for new types of centrally acting muscle relaxants. The derivtives with an alkylaminopropyl moiety exhibited muscle relaxant activity comparable to that of tolperisone (1), but this activity was accompanied by strong general central nervous system (CNS) depressant and slight anticonvulsant activities.On the other hand, the derivative with a propanamide moiety exhibited a favorable pharmacological profile for a selective muscle relaxant with little CNS depressant action.

The steric course of the reaction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase from Escherichia coli was investigated. trans-2-[6,7,8-2H7]Octenoyl-CoA was synthesized as a substrate and incubated with partially purified trans-2-enoyl-CoA reductase in the presence of 4R- or 4S-[4-2H1]NADPH. The deuterium-labeled octenoyl-CoA was also incubated with the reductase in the presence of NADPH in 2H2O. Aliquots of octanoic acids formed were analyzed, after esterification, by gas chromatography-mass spectrometry (GC-MS) to demonstrate that the pro-4R hydrogen of NADPH was incorporated into the C-3 position of octenoyl-CoA and that hydrogen from water was introduced into the C-2 position of octenoyl-CoA. The remaining portions of octanoic acids isolated from the incubation mixtures were converted to their CoA esters by the action of acyl-CoA synthetase, and they were dehydrogenated by treatment with acyl-CoA oxidase, which had previously been shown to catalyze the anti-elimination of the pro-2R and pro-3R hydrogen atoms of acyl-CoA. The deuterium contents of the products were also analyzed by GC-MS, and the results indicated that the reduction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase occurred by an anti-addition of hydrogen via a 2-Re, 3-Re attack on the trans-double bond of the substrate.

Crude extract of Escherichia coli was found to elongate medium chain acyl-CoA primers. The reaction products were fatty acids one or two C2 units longer than the primer. Acetyl-CoA acted as the condensing unit in this reaction, while malonyl-CoA did not. The optimal pH for the reaction was 5.0 in 0.1 M citrate-phosphate buffer. NADH was the predominant electron donor for the incorporation of acetyl-CoA into fatty acids, and NADPH was one-third as effective as NADH at pH 5.0. Acyl carrier protein and cerulenin had no effect on the acetyl-CoA incorporation into the chain elongation products. Acyl-CoA compounds with medium carbon chain lengths proved to be the best as primers, and the maximum incorporation was observed with octanoyl-CoA. N-Ethylmaleimide and p-hydroxymercuribenzoate blocked the chain elongation reaction by inhibiting either condensation or 3-ketoacyl reduction.

It is demonstrated that 3-alkenoyl-CoA is the product of the enzymic reduction catalyzed by 2,4-dienoyl-CoA reductase with the highly purified enzyme preparation from beef liver mitochondria. Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase from beef liver was investigated. When trans-2, trans-4-decadienoyl-CoA was incubated with the reductase in the presence of 4R-[4-2H1]NADPH and H2O, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to pyrrolidine amides of fatty acids. When the substrate was incubated with the enzyme in the presence of 4S-[4-2H1]NADPH and H2O, one deuterium atom was incorporated into the product, 3-decenoate, at the C-5 position. In the case of 3-decenoate enzymatically prepared in the presence of NADPH and 2H2O, two deuterium atoms were incorporated into the product at the C-2 position. These results indicate that the reaction catalyzed by 2,4-dienoyl-CoA reductase from beef liver is a unique example of 1,4-addition of hydrogen to a 1,3-diene system conjugated with a carbonyl group of a thioester in biochemical fields. Chain length specificity of the reductase, isotope effects on reaction rates and competitive inhibitors are also discussed.

Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli (E. coli) was investigated. When trans-2,cis-4-decadienoyl-CoA was incubated with 4R- or 4S-[4-2H1]NADPH in the presence of purified 2,4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide. On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O, two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position. The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group. Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.

Two fractions containing NADPH-enoyl CoA reductase activities were isolated from crude extracts of Candida albicans. One fraction (Type-I) having larger molecular weight utilizes 5-hydroxyundec-cis-2-enoyl CoA, oct-cis-2-enoyl CoA and oct-trans-2-enoyl CoA as substrates with Km values of 2.5×10-6M, 1.1×10-6M and 5.0×10-7M, respectively, while another fraction (Type-II) having smaller molecular weight utilizes these substrates with Km values of 3.0×10-6M, 5.3×10-5M and 1.0×10-5M, respectively. This indicates that there exist comparable differences between these two in their affinities especially for the latter two substrates. Some characterizations, such as, effects of pH and of heat treatment on the activities of these reductase preparations were also investigated.

An enzyme, β-hydroxybutyryl acyl carrier protein (ACP) dehydrase, which catalyzes the reversible dehydration of short chain β-hydroxyacyl-ACP to the corresponding α, β-unsaturated acyl-ACP derivatives has been isolated from extracts of Escherichia coli. Dehydrase preparations of specific activity of 5000 mµmoles per min per mg of protein were homogeneous as assessed by ultracentrifugal analysis and by polyacrylamide disc gel electrophoresis. The enzyme has an estimated molecular weight of 26,000. It is relatively heat-stable and is active over a broad pH range with maximal activity between pH 7.5 and 8.5. The dehydrase has a functional —SH group and can be readily inhibited by —SH-binding reagents such as N-ethylmaleimide and iodoacetamide. The dehydrase reaction is readily reversible and the equilibrium constant for the dehydration of β-hydroxybutyryl-ACP is estimated to be 19 m. The β-hydroxybutyryl-ACP dehydrase is active on ACP thioesters but inactive on the coenzyme A derivatives. The enzyme is specific for short chain length β-hydroxyacyl-ACP derivatives (C4 to C8). The estimated maximal rates of hydration of crotonyl-ACP, 2-hexenoyl-ACP, and 2-octenoyl-ACP are 4100, 2300, and 200 mµmoles per min per mg, respectively. The enzyme is inactive on 2-decenoyl-ACP. This limited chain length specificity of the β-hydroxybutyryl-ACP dehydrase is responsible for the accumulation of β-hydroxydecanoyl-ACP in a reconstituted fatty acid-synthesizing system comprised of highly purified preparations of malonyl-CoA-ACP transacylase, acyl-malonyl-ACP condensing enzyme, β-ketoacyl-ACP reductase, β-hydroxybutyryl-ACP dehydrase, and enoyl-ACP reductase. The β-hydroxy-decanoyl-ACP thus synthesized is readily converted to either saturated or unsaturated fatty acids by the action of protein fractions which contain at least two long chain βhydroxyacyl-ACP dehydrases.

An enzyme fraction which catalyzes the reduction of trans-2-enoyl-coenzyme A (trans-2-enoyl-CoA) to the corresponding saturated acyl-CoA derivative was separated from Escherichia coli (E. coli) extracts. The enzyme utilized reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a sole electron donor for the reduction of substrates, and it acted on acyl-CoA substrates, but not on acyl-carrier protein esters. Therefore, the name NADPH-dependent trans-2-enoyl-CoA reductase was proposed for this enzyme. The reductase was stable at around pH 7-8, and was active over a wide pH range between 6 and 9. It had a functional thiol group and was readily inhibited by thiol reagents such as p-hydroxymercuribenzoic acid, and by cupric and some other metal ions. The enzyme was induced when E. coli cells were cultured in a medium containing oleic acid. These results suggest that this reductase does not participate in the de novo synthesis of fatty acids but is a new enzyme, although its physiological role is uncertain at the present stage.

Conformationally restricted analogs of muscle-relaxant 3-amino-2-methyl-N- (3-phenyl-5-isoxazolyl) propanamides (1) and 5- (3-aminopropylamino) -3-phenylisoxazoles (2) were prepared, and their muscle-relaxant and other pharmacological activities were tested and compared with those of the corresponding acyclic derivatives. 7- (3-Diethylamino-2-methylpropanoyl) -3-phenyl-4, 5, 6, 7-tetrahydroisoxazolo [5, 4-b] pyridine (8) exhibited muscle-relaxant and anticonvulsant activities comparable with those of corresponding acyclic derivatives, i.e. 3-diethylamino-2-methyl-N- (3-phenyl-5-isoxazolyl) propanamides (1e-g), but other types of compounds showed decreased activities. The preferred conformation of the present isoxazole derivatives for muscle-relaxant activity is discussed.7-Benzyl-6-methyl-3-phenyl-4-pyrrolidino-4, 5, 6, 7-tetrahydroisoxazolo [5, 4-b] pyridine (3d) showed moderate central nervous system-depressant activity.

The stereochemical mechanism of the reduction of cis-2-octenoyl-coenzyme A (-CoA) catalyzed by cis-2-enoyl-CoA reductase from E. coli was studied. cis-2-[7, 8-2H5] Octenoyl-CoA was incubated with partially purified cis-2-enoyl-CoA reductase in the presence of 4R or 4S-[4-2H1] nicotinamide adenine dinucleotide phosphate (NADPH). The octenoyl-CoA was also incubated with the enzyme in the presence of NADPH in 2H2O. Octanoic acids synthesized were isolated and analyzed by gas chromatography-mass spectrometry (GC-MS) to examine the localization of deuterium atoms in the molecule. The octanoic acids isolated from the incubation mixtures were converted to their CoA esters, and then they were dehydrogenated by the action of acyl-CoA oxidase, which had previously been shown to catalyze the anti-elimination of the pro-2R and pro-3R hydrogens of acyl-CoA. The resulting products, trans-2-octenoyl-CoAs, were converted to methyl esters and their deuterium contents at the C-2 or C-3 position were also analyzed by GC-MS. The results suggested the following stereochemical features of the reaction catalyzed by cis-2-enoyl-CoA reductase. 1) pro-4R Hydrogen of NADPH was incorporated into the C-3 position of octenoyl-CoA. 2) Hydrogen from the medium was introduced into the C-2 position of octenoyl-CoA. 3) The reduction occurred by an anti-addition of hydrogen via a 2-Si, 3-Re attack on the cis-double bond.

The mode of neocarzinostatin-chromophore (NCS-chr)-apo-neocarzinostatin (apo-NCS) interaction in neocarzinostatin (NCS) complex has been described. The NCS-chr release from the NCS complex in the presence of various reagents, which destroy the high-order structure of the protein under various pH conditions, was examined. We found that (i) sodium dodecylsulfate, Nonidet P 40, 8 M urea, and 2-propanol did release NCS-chr from NCS, (ii) no NCS-chr release is detected below pH 7, but it is enhanced at high pH and (iii) beta-naphthol as a model of naphthalenecarboxylic acid derivative and D-galactosamine as a model of N-methylfucosamine of NCS-chr did release NCS-chr from NCS. These observations indicate that the binding of NCS-chr to apo-NCS may be due to not only ionic interaction between the acidic side chain of apo-NCS and the basic center of an aminosugar moiety of NCS-chr but also hydrophobic interaction between the hydrophobic amino acids of apo-NCS and hydrophobic moieties of NCS-chr. Apo-NCS is a very hydrophilic protein, since it has an high hydrophilic amino acid content. So, local hydrophobicity, local hydrophilicity and secondary structure of apo-NCS were predicted. Hydrophobic residues of apo-NCS predominantly located in beta-sheet structures near the carboxyl-terminus. These predictions are in good agreement with the results suggesting that NCS-chr bound carboxyl-terminal-43-peptide of apo-NCS in our previous result.

Seventeen patients with various grades of kidney function were injected each with cefsulodin (CFS) 1 g dissolved in 20 ml of saline. Serum and urine concentrations of CFS were determined using high performance liquid chromatography up to 24 hours after administration. Pharmacokinetic analysis was done using a two-compartment model. The results were discussed by comparing creatinine clearance (Ccr) values divided into 4 groups: greater than or equal to 70 ml/min (group I), 50-less than 70 ml/min (group II), 30-less than 50 ml/min (group III), and less than 30 ml/min (group IV). A delay in the disappearance of CFS from the blood was observed with a decrease in Ccr. Half-lives of CFS in blood (T 1/2 beta) were 1.03 hours (group I), 2.09 hours (group II), 3.44 hours (group III), and 4.52 hours (group IV). Serum clearance (Cls) and Ccr were found to be related with an equation: Cls = 1.60 x Ccr + 7.70. The correlation coefficient (r) was 0.881. Ccr and T 1/2 beta were found to be related with an equation: T 1/2 beta = 31.27 x Ccr-0.688, and the area under the curve (AUC) was found to be related to Ccr with AUC = 4,226 x Ccr-0.81. Urinary excretion rates up to 24 hours after administration were 84.4% (group I), 69.1% (group II), 67.5% (group III), and 56.5% (group IV). This means that CFS is excreted in urine with a relatively high recovery even in the case of low Ccr less than 30 ml/min.

N-Substituted 3-amino-2-methyl-N-(3-phenyl-5-isoxazolyl)propanamide derivatives (5a-w) were prepared, and their muscle relaxant and anticonvulsant activities were evaluatd. Among the compounds with high potency, 3-diethylamino-2, N-dimethyl-N-(3-phenyl-5-isoxazolyl)propanamide (5e) was found to possess selective muscle relaxant and anticonvulsant activities. Compounds with carbon side chains other than the 2-methylpropanamide moiety did not give increased muscle relaxant activity. Optical isomers of 5e were prepared from metyl (+)-and (-)-3-diethylamino-2-methylpropionates (15a, b), and the (+)-isomer (5x)b was found to be twice as potent as the (-)-isomer (5y). The structure-activity relationship was studied with emphasis on the effects of the 3-amino moiety and of the substituent on the isoxazolyl 5-amino group. A good quadratic correlation equation was found between hydrophobicity and muscle relaxant activity.

The activity of acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli was enhanced when the organism was grown on oleic acid as the sole carbon source, but not detected when grown on glucose. Antibodies raised against fatty acid oxidation complex of E. coli inhibited both the reaction catalyzed by crotonase and the chain elongation in a similar manner, showing that the oxidation complex participates in the chain elongation. The activities of condensation and the activities of NADH- and NADPH-dependent 3-ketoacyl reduction in the cell-free extract were precipitated by antibodies to the complex in parallel with those of 3-ketoacyl-CoA thiolase and crotonase. These results together with the presence of NADPH-dependent trans-2-enoyl-CoA reductase in E. coli (Mizugaki, et al. (1982) Chem. Pharm. Bull. 30, 2503-2511) indicate that the acetyl-CoA-dependent chain elongation of fatty acids in E. coli occurs by the reversal of fatty acid oxidation other than the step of enoyl reduction.