Our analysis of serum cholinesterase (ChE) isoenzyme pattern and serum cholinesterase activity using Wistar rats disclosed the following sex- and age-related differences:
1. The ChE isoenzymes of the Wistar rats were found to consist of six distinct fractions. There were no detectable sex- or age-related variation in the number of ChE fractions.
2. In the female rats, band 6 was present in the highest proportion, 73% (relative concentration) of the sum of six bands, followed by band 5 that accounted for 19%. Bands 6 and 5 were regarded as the main bands and bands 3, 2, 1, and 4 (in descending order) were considered as sub-bands. In the male rats, the proportion of band 5 was almost equal to that of band 6 for the six week old group. The proportion of band 5 (70%) was much higher than that of band 6 (17%) for the twelve week old group. Thus, a sex-related difference was detected in the proportion of the bands, as well as an agerelated difference.
For the determination of the rat serum ChE activity, Ellman's method, which utilizes acetylthiocholine (AcSCh) as a substrate, proved to be more suitable than the method which uses acetylcholine (ACh), because the ChE activity determined using the latter method can be less than that determined by the former method, particularly for female rats during their growth phase.

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Tryptophan-poor α₁-glycoprotein was initially isolated by Haupt and Heide in 1964, and designated as α₁-T glycoprotein (α₁-T). α₁-T was originally isolated from pooled human serum, with 5 steps of chromatography, and improved by introducing affinity chromatography conjugated with anti α₁-T monoclonal antibody.
The molecular weight of purified α₁-T was about 77, 500 daltons determined by SDS-PAGE.
Amino acid composition were determined and was almost identical to that obtained by Schwick. Partial amino acid sequence was-Glu-Ser-Leu-Leu-Asn-His-Phe-Leu-Tyr-Glu……which did not show any homology to that reported with all existing proteins.
Anti α₁-T rabbit serum and anti α₁-T monoclonal antibody were prepared with purified α₁-T.

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We examined two families with silent type hypocholinesterasemia, by using electrophoretic analysis and immunological method.
One case had trace amount of normal cholinesterase (ChE; EC 3.1.1.8.) activity. By immunological method, production of ChE protein was observed in the case. A part of the protein was seemed to show ChE activity. The case was suspected to be caused by a disturbance of polymerization, because C4 band was weak by electrophoretic analysis. Another case had neither ChE activity nor production of ChE protein. The former was homozygote of silent gene type II, and the latter, homozygote of silent gene type I.
Therefore, heterogeneity of the silent gene is very rich in Japan and variation is demonstrated to be in some steps of ChE production.

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Various different stainings were tested with cellulose acetate membrane as a supporting medium, in order to study their sensitivities for staining protein fractions at very low concentrations. The pigment Acid violet 17 was found to be most sensitive, requiring only a sample size of 1μl for a sample with a total protein concentration of 200mg/dl and 4μl for a total protein concentration of 50mg/dl. Acid violet 17 staining was approximately 10 times more sensitive than Ponceau 3R staining, and since cerebrospinal fluid may be analysed in the unconcentrated form at this sensitivity, it promises to be clinically useful. Acid violet 17 staining is easy to perform, and gave advantages over other stainings in terms of sensitivity, rapidity, and economy, hence we believe Acid violet 17 staining provides a very suitable method for routine analysis.

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The nonenzymatic glucosylation reaction occurs in various proteins of the body, particularly in diabetic subjects. Glucosylated human albumin was prepared by incubating albumin with glucose in the presence or absence of sodium cyanoborohydride (NaCNBH₃) for 2 weeks at 37°C. We prepared a newly boronate affinity chromatography system by connecting 3-aminophenyl boronic acid hemisulfate with CNBr-activated Sepharose 4B. Using this system, synthetic glucosylated albumin, normal serumm and diabetic were applied to this Boronate Sepharose column. Pure glucosylated protein was separated from them. Glucosylated albumin levels to the total albumin were 49% in the synthetic nonreduced glucosylated albumin. Glucosylated protein levels to the total protein were determined in 5 of the diabetic subjects and ranged from 10.4% to 15.7% (mean±SD 11.2±2.0). Glucosylated protein levels ranged from 5.6 to 8.0 (mean±SD 6.9±0.9) in 5 of the normal subjects. Glucosylated protein values were significantly greater in diabetic subjects than in normal subjects. The reduced glucosylated albumin moved more rapidly than nonglucosylated albumin and nonreduced glucosylated albumin on agarose electrophoresis. However, the reduced glucosylated albumin on polyacrylamide electrophoresis moved a little more rapidly than the other albumins.
A male rabbit was immunized with human reduced glucosylated albumin. On day 120 blood was obtained for our studies. To document the presence of antibodies, we used the Ouchterlony method and confirmed an antibody against reduced glucosylated albumin.

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Colloidal gold was used to detect extremely small amount of proteins electrotransferred on nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Clearly deep wine-red bands of proteins were obtained by using 0.002∼0.005% colloidal gold solution stabilized with Tween 20. The sensitivity was in the same range as silver staining of polyacrylamide gel and enough to detect nanogram order of proteins applied on the gel.

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