The rat submaxillary gland kallikrein (RSGK)(EC 3.4.21.35) was further evaluated. The enzyme was purified to a homogeneous state following the combination of ion exchange chromatographies and gel filtrations. The final kallikrein preparation had a specific activity of 20.4 μmol/min/A₂₈₀ for N-a-tosyl-L-arginine methyl ester (Tos-Arg-Me) and 132 kallikrein unit (KU) /A₂₈₀ in a vasodilatation assay, representing a 140 fold purification with an overall yield of 25% of Tos-Arg-Me esterolytic activity from the initial extract. The molecular weight of present enzyme was estimated to be 3.8×10⁴ daltons by Sephadex G-100 gel filtration and SDS gel electrophoresis. The optimum pH for Tos-Arg-Me esterolysis was between 8.5 to 9.5. The RSGK had the esterolytic and amidolytic activities showing wide response profiles against the substrates of synthetic arginine and lysine derivatives. It was found that the N-α-benzoyl-L-arginine methyl ester (Bz-Arg-Me) was most effective one for the RSGK, in the examined. Among the various proteinase inhibitors tested, aprotinin was most inhibitory, while α₁-antitrypsin and antithrombin III was less.

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A radioimmunoassay has recently been developed for the quantitation of opioid peptides such as methionine-enkephalin and leucine-enkephalin isolated from the adrenal medulla. However, the current assay system still has some problems including immunological cross-reactivity which is often encountered, as well as proteolytic degradation of the peptides during the extraction procedure from the tissue.We have performed a series of basic experiments to improve the assay system of the opioid peptides present in adrenal medulla by overcoming the problems described above, and applied the improved method to an in vitro analysis of enkephalin secretion from the adrenal medulla.
The present methionine-enkephalin assay system proved to be satisfactory with respect to intra-assay variation and recovery ratio, with minimal cross-reactivity with leucine-enkephalin. However, leucine-enkephalin assay system showed significant degree of cross-reactivity with methionine-enkephalin, which could be reduced from 20% to as low as 1% by oxydizing the samples with hydrogen peroxide before the test.
We have also shown that spontaneous degradation of enkephalins during the extraction from a tissue could be prevented by the inclusion of peptidase inhibitors such as amastatin, puromycin or phosphoramidon in the extraction procedure. Boiling of tissue homogenate in acetic acid also proved useful in preventing an eventual loss of enkephalin immuno-reactivity. On the contrary, extraction with sucrose solution or hydrochloric acid resulted in low immunoreactivity.
Finally, we examined the kinetics of opioid secretion from the adrenal medulla in vitro, using our improved assay method. A continuous flow incubation system was developed for this purpose, in which secretory response of adrenal medulla to nicotine was characterized by serial fluorimetric assay of catecholamines and a radioimmunoassay of enkephalins in the effluent medium. During the course of perifusion, the initial release of a large volume of catecholamines and enkephalins decreased in one hour and a half to the baseline level and remained at the level thereafter. In 2 hours after the start of perifusion, both catecholamines and enkephalins output rose abruptly after the addition of nicotine and returned promptly to the baseline after its withdrawal. Thus, we have shown that enkephalins were co-released from the adrenal medulla with catecholamines in response to nicotine by perifusion experiment.

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A new method for the analysis of urinary magnesium using hexokinase (EC 2.7. 1.1) or glucokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was established. The method is based on the determination of absorbance at 340 nm for NADPH formed by the overall reaction of hexokinase or glucokinase activated by Mg²⁺ and glucose-6-phosphate dehydrogenase. The reaction rate of hexokinase or glucokinase is proportional to the amount of Mg·ATP^2- complex. This simple, enzymatic method does not require an expensive instrumentation, but it provides the results in excellent agreement with those obtained by atomic absorption spectrometry. Since the present method gives linearity over a wider range on the standard curve of magnesium than the currently used chelator method does, it permits direct determination on urine samples without previous dilution.

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Ascorbic acid (AH₂) is well known to give false positive errors in the determination of hydrogen peroxide (H₂O₂) with the catalase-Hantzsch reaction which involves the formation of formaldehyde from H₂O₂ and methanol, followed by the color development based on the condensation reaction of formaldehyde formed and acetylaceton added in the presence of ammonium ion.
Present study was undertaken to elucidate a possible mechanism of the AH₂ interference in the H₂O₂ determination. Dehydro-ascorbic acid (DA) was prepared by use of immobilized ascorbate oxidase-tube. The oxidation-reduction relationship between AH₂ and DA was investigated with spectral analysis and using the Hantzsch reaction and aldehyde dehydrogenase reaction.
1.AH₂ continuously increases the color development of the catalase-Hantzsch reaction in the absence of H₂O₂, suggesting the formation of H₂O₂ or formaldehyde in the reaction mixture.
2.AH₂ is oxidized by a sparing amount of oxygen and Cu²⁺ to form H₂O₂ and DA.
3.DA formed reacts with methanol even in the absence of catalase to regenerate AH₂ and formaldehyde. H₂O₂ reacts with methanol in the presence of catalase to form formaldehyde. The formaldehyde increases the color development of the Hantzsch reaction.
4.AH₂ regenerating system (AH₂, O2 and methanol) continuosly supplies H₂O₂ and formaldehyde which are used as substrates of the catalase-Hantzsch reaction and then increase the color development. The coupled reaction of DA reduction (DA to AH₂) and methanol oxidation (methanol to formaldehyde) seems to play a most important role in AH₂ interference.

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Since a simple method for assay of polyamines in urine has been developed, urinary polyamines are widely measured in clinical laboratories. Urinary polyamine levels, however, are known to be affected by contamination with some kind of enterocolic bacteria, and by the methods of collecting urine. We have recently reported that sodium azide is a useful preserver to collect 24-h urine samples for polyamine assay. Collecting urine for 24 h, however, is usually a lot of trouble for out-patients and this prevents popularization of urinary polyamine assay in clinical medicine. We report here that the total polyamine values in 2-h urine samples, but not the polyamine/creatinine ratios in freshly voided urine samples, are correlated well with the polyamine values excreted during 24 h in urine for both healthy subjects and patients {y (μmol/2h) =0.09× (μEmol/24h) + 0.0874, r=0.906}, and that urinary excretion of cadaverine fluctuates remarkably during a day in some persons while other kind of polyamines does not.
The remarkable difference in urinary polyamine excretion among healthy individuals is mainly ascribed to the changes in cadaverine excretion. The reason why this marked difference is mainly occured in cadaverine remains to be clarified.

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Basic human urinary arginine esterase (BHUAE) and basic human seminal plasma arginine esterase (BHSAE) were separated from the male urine and seminal plasma, respectively. The percentage of the content level of BHUAE was calculated to be about 1 to 3% of the total N-α-tosyl-L-arginine methyl ester (Tos-Arg-Me) esterolytic activity in the male urine. The esterolytic activities of BHUAE and BHSAE towards synthetic amino acid ester derivatives were measured and substrate specificity of both enzymes was not agreed.

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It was revealed that the immunochemical isolation method for GOT isoenzymes, using anti pig c-GOT antibody, could be applicable for dogs, cats, rats, chickens and mice in addition to pigs and human. Mitochondrial-GOT/total-GOT (m-GOT/t-GOT) ratios were not necessarily close to one in various organs of each animal and furthermore the distributive differences among the corresponding organs of those species were also evidently recognized by this method.

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The effect of thiamin on excretion of lead into bile was investigeted using isolated rat liver perfusion. The amount of lead excreted into bile was increased by addition of thiamin to the perfusate containing lead acetate. Furthermore, the excreted lead in bile was fractionated by gel chromatography on Sephadex G-75. Lead in the bile was detected in three fractions which corresponded to a high molecular weight and two low molecular weight fractions.
These results lsuggested that the excretion of lead into bile was enhanced by thiamin and that lead taken up in the liver was secreted into bile in which lead might be bound to certain proteins and low molecular substances such as glutathione and thiamin

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