p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme

p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.     

Effect of DN-1417, a thyrotropin releasing hormone analog, on dopaminergic neurons in rat brain

Acute and chronic effects of γ-butyrolactone-γ-carbonyl-histidyl-prolinamide (DN-1417) were investigated on motor activity, dopamine (DA) metabolites and DA receptors in various brain regions of rats. The motor activity, as measured with Automex recorder, was enhanced after a single injection with DN-1417 (20 mg/kg, IP), and the motor stimulating action persisted during 21 daily injections. Acute DN-1417 elevated both homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in 7 brain regions, prefrontal cortex polar, medial and lateral fields, nucleus accumbens, olfactory tubercles, amygdala and striatum. After chronic treatment for 7 days, the acute effect of DN-1417 on DA metabolites disappeared in all regions except for the striatum in which DN-1417 still increased HVA and DOPAC. The response of striatal DA metabolites was also observed after chronic treatment for 21 days. Chronic DN-1417 produced no significant change in ³H-spiperone binding in the prefrontal cortex, nucleus accumbens, olfactory tubercles and striatum, while striatal ³H-DA binding displaced by 30 nM spiperone was enhanced after chronic treatment. These results indicate that DN-1417 interacts with mesocortical, mesolimbic and nigrostriatal DA systems in the different modes of action. The lack of tolerance to motor hyperactivity, however, raises the question as to whether DN-1417-induced hyperactivity may be mediated by the activation of mesolimbic DA neurons. The involvement of nigrostriatal neurons in DN-1417-induced motor hyperactivity is suggested.     

Selective enhancement in striatal [H]nitrendipine binding following chronic treatment with morphine

Two parameters of Ca²⁺ dynamics in brain preparations (⁴⁵Ca-uptake to slices and [³H]nitrendipine binding to membrane fractions) were measured in naive and chronic morphine-administered rats. While morphine did not have any effect on ⁴⁵Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K⁺-stimulated ⁴⁵Ca-uptake to slices. Furthermore, the effect of morphine was antagonized by naloxone. Inhibition of K⁺-stimulated ⁴⁵Ca-uptake to striatal slices by morphine was not observed in preparations obtained from chronic morphine-administered rats (6 mg/kg/b.i.d./7 days). In membrane fractions, [³H]nitrendipine binding increased by 34% in striatum following chronic morphine treatment, whereas no change was observed in the cortex and hippocampus. The results will be discussed in relation to the phenomena underlying chronic morphine administration.     

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD⁺ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD⁺ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

A versatile bacterial enzyme: l-methionine γ-lyase

The properties and application of l-methionine γ-lyase [methioninase, l-methionine methanethiol-lyase (deaminating), EC 4.4.1.11], a pyridoxal 5′-phosphate enzyme, purified from Pseudomonas putida and Aeromonas sp. are presented. The enzyme has multicatalytic functions: it catalyses α,γ-elimination and γ-replacement reactions of l-methionine and its analogues (e.g. ethionine, homocysteine, O-acetylhomoserine and selenomethionine), α,β-elimination and β-replacement reactions of l-cysteine and its analogues (e.g. S-methylcysteine, O-acetylserine and Se-methylselenocysteine), deamination and γ-addition of vinylglycine, and deuterium labelling at the α and β positions of l-methionine and other straight-chain l-amino acids. These reactions are applicable to the synthesis of various optically active sulphur and selenium amino acids, preparation of deuterium or tritium labelled l-amino acids, and determination of sulphur amino acids. In addition, the enzyme shows potent anti-neoplastic activity.     

Screening of thermostable leucine and alanine dehydrogenases in thermophilicBacillus strains

Summary Screening of leucine and alanine dehydrogenases in thermophilicBacillus strains was carried out to develop their utilization for industrial and analytical catalysts. Out of the 28 thermophilic strains tested, four strains,Bacillus sp. DSM 405, 730 and 1521, andB. sphaericus DSM 462, abundantly produce both the enzymes. Both the enzyme activities in these thermophiles are enhanced by addition of the substrates to a polypeptone medium.     

Restriction fragment length polymorphism detected by human salivary amylase cDNA

Summary The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7kb and 6.5kb fragment alleles, are 0.55 and 0.45, respectively.     

A genetically restricted suppressor factor that requires interaction with two distinct targets

We have previously described a genetically restricted suppressor factor (TsF3) that suppresses the terminal phases of the contact sensitivity response. The activity of TsF3 is restricted by genes in the H-2 (I-J) and Igh complexes. This report analyzes the mechanisms responsible for these genetic restrictions. One cellular target of TsF3 is an I-J-bearing antigen-presenting cell population that is sensitive to low doses of cyclophosphamide. To elicit suppression I-J homology is required between this antigen-presenting cell population and the TsF3 donor. In contrast, the Igh-linked genetic restriction exists between TsF3 and an unprimed cell population present in the recipient. These findings suggest that under these experimental conditions TsF3 acts by bridging the APC with cells of the host. Finally, we demonstrated that nonspecific bystander or cognate suppression can be mediated by TsF3, provided specific antigen is present in the site of the ongoing T cell response.