In urine of diabetics, a significantly great inhibitory activity of glyceraldehyde 3-phosphate dehydrogenase (GAP-Dehyd) was observed compared with that of normal subjects. The 0.2 ml of urine from 41 patients with diabetes mellitus inhibited 0.5 units of GAP-Dehyd by 27.2 +/- 3.0% (mean +/- S.E.M.), while that from 17 normal volunteers inhibited only by 9.0 +/- 1.0% (P less than 0.05). This inhibitory substance was extracted by 90% ethanol from diabetic urine and partially purified by anion exchange chromatography using Dowex-1 (HCOO type). The molecular weight of this substance was confirmed to be 100--300 daltons by an analysis on Biogel P-4 gel filtration chromatography. And analysis by thin layer chromatography using silicagel plate showed that this inhibitor was a ninhydrin reactive substance which has not been reported previously. From the above facts, it was assumed that the inhibitory substance of GAP-Dehyd in urine of diabetics was a new acidic compound of low molecular weight containing an amino residue in the molecule.</p

Kawanishi, Koichi

Terato, Kuniaki

Yamamoto, Shinro

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 32, Issue 5 1978 Article 3OCTOBER 1978An inhibitory substance of glyceraldehyde3-phosphate dehydrogenase in urine ofdiabetic patientsKuniaki Terato∗ Koichi Kawanishi†Shinro Yamamoto‡∗Okayama University,†Okayama University,‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.An inhibitory substance of glyceraldehyde3-phosphate dehydrogenase in urine ofdiabetic patients∗Kuniaki Terato, Koichi Kawanishi, and Shinro YamamotoAbstractIn urine of diabetics, a significantly great inhibitory activity of glyceraldehyde 3-phosphatedehydrogenase (GAP-Dehyd) was observed compared with that of normal subjects. The 0.2 ml ofurine from 41 patients with diabetes mellitus inhibited 0.5 units of GAP-Dehyd by 27.2 +/- 3.0%(mean +/- S.E.M.), while that from 17 normal volunteers inhibited only by 9.0 +/- 1.0% (P less than0.05). This inhibitory substance was extracted by 90% ethanol from diabetic urine and partiallypurified by anion exchange chromatography using Dowex-1 (HCOO type). The molecular weightof this substance was confirmed to be 100–300 daltons by an analysis on Biogel P-4 gel filtrationchromatography. And analysis by thin layer chromatography using silicagel plate showed that thisinhibitor was a ninhydrin reactive substance which has not been reported previously. From theabove facts, it was assumed that the inhibitory substance of GAP-Dehyd in urine of diabetics wasa new acidic compound of low molecular weight containing an amino residue in the molecule.KEYWORDS: glyceraldehyde 3-phosphate dehydrogenase, inhibitor, diabetes mellitus, urine∗PMID: 153095 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 32, (5), 337-342 (1979)AN INHIBITORY SUBSTANCE OF GLYCERALDEHYDE3-PHOSPHATE DEHYDROGENASE IN URINEOF DIABETIC PATIENTSKuniaki TERATO, Koichi KAW ANISHIand Shinro YAMAMOTOThird Department of Internal Medicine, Okayama University Medical School,Okayama 700, Japan (Director: Prof. T. Ufuji)Received May 29, 1978.Abstract. In urine of diabetics, a significantly great inhibitory activ-ity of glyceraldehyde 3-phosphate dehydrogenase (GAP-Dehyd) wasobserved compared with that of normal subjects. The 0.2 ml of urinefrom 41 patients with diabetes mellitus inhibited 0.5 units of GAP-Dehydby 27.2±3.0% (mean±S.E. M.), while that from 17 normal volunteersinhibited only by 9.0 ± 1.0% (P<0.05). This inhibitory substance wasextracted by 90% ethanol from diabetic urine and partially purified byanion exchange chromatography using Dowex-l (HCOO type). Themolecular weight of this substance was confirmed to be 100-300 daltonsby an analysis on Biogel P-4 gel filtration chromatography. And analy-sis by thin layer chromatography using silicagel plate showed that thisinhibitor was a ninhydrin reactive substance which has not been re-ported previously. From the above facts, it was assumed that the in-hibitory substance of GAP-Dehyd in urine of diabetics was a new acidiccompound of low molecular weight containing an amino residue inthe molecule.Key words: glyceraldehyde 3-phosphate dehydrogenase, inhibitor,diabetes mellitus, urineIt was reported that an inhibitory substance(s) of glyceraldehyde 3-phos-phate dehydrogenase (GAP-Dehyd) was present in plasma of diabetics (1,2).But the chemical identification of this substance has not been performed yet, andits originating organ and role in abnormal carbohydrate metabolism in diabetesmellitus are not fully understood at present.Bornstein and his co-workers reported that this inhibitory substance was apolypeptide derived from the pituitary gland (3, 4), i.e. a peptide fragment ofgrowth hormone (5). However, Schwartz and Turfus (2) have recently showedthat a nonpeptide inhibitory substance of GAP-Dehyd with lower molecularweight was present in plasma of diabetics, and the peptide of growth hormonewas not detected.In an attempt to verify the presence of an inhibitory substance of GAP-3371Terato et al.: An inhibitory substance of glyceraldehyde 3-phosphateProduced by The Berkeley Electronic Press, 1978338 K. TERATO et aI.Dehyd in diabetes mellitus, the inhibitory activity of unne of diabetics wasassayed.MATERIALS AND METHODSUrine samples. Urine samples were collected at 9-12 a.m. from 41 patients(aged 20-67) with diabetes mellitus who visited and were hospitalized at OkayamaUniversity Hospital. The samples were stored at -20°C until use. Control urinesamples were collected at 10-11 a.m. from 17 normal volunteers (aged 20-47).Most of the subjects were not fasted.Reagents. Crystalline rabbit muscle GAP-Dehyd (EC. 1. 2. 1. 12), ,9-NAD andD-glyceraldehyde 3-phosphate (GAP) were purchased from Sigma Chemical Co.,Ltd., U.S.A.. Ninhydrin solution was purchased from Tokyo Kasei Co., Ltd., andpetroleum pitch charcoal with a spherical granule (BAC-LQ) was supplied byKureha Kagaku Co., Ltd., Tokyo.Assay of inhibitory activity of GAP-Dehyd. The assay of inhibitory activity ofGAP-Dehyd in urine was performed according to the method of Schwartz andTurfus (2). The reaction mixture contained: 2.50 ml of 0.1 M pyrophosphatebuffer containing 0.02 M EDTA, pH 8.45; 0.10 ml of 1.02 X 10-2 M p-NAD; 0.10ml of GAP-Dehyd solution (5 units/ml); and 0.20 ml of urine from the patients.The reaction was started by adding 0.10 ml of 1.02 X 10-2 M GAP and the opticaldensity at 340 nm was measured at 25 ± 3°C for 5 min using a Hitachi Spectro-photometer Model 124. The inhibitory activity (%) was calculated as follows,[(0. D. cont-O. D. test)/O. D. cont] X 100.Determination of the molecular weight. The molecular weight of the inhibitorysubstance of GAP-Dehyd was determined by gel filtration chromatography onBiogel P-4 (column 1.5 X 82 em) using vitamin B12 (mol. wt. 1350) and CoC12 (mol.wt. 130) as standard. 0.05 M acetic acid was used as an effluent at a flow rate of10 ml/h, and 3 ml fractions were collected.Purification of the GAP-Dehyd inhibitory substance. The GAP-Dehyd inhibitorysubstance was extracted with ethanol (final 90%) from urine (0.5-lliter) of dia-betics, and dried in vacuo at below 50°C. The dried residue was dissolved in200 ml of distilled water, and then 50 g of Dowex-50 (H+ type) and 50 g of acti-vated charcoal was added to it and stirred for 30 min at 4°C. After removing theresin and charcoal by filtration on a glass filter (No.3), the solution was adjustedto pH 8 by adding triethylamine and applied to a column of Dowex-l (HCOO-type) (3x35cm). Elution was carried out at first with 300ml of 10% ethanol andthen with a linear gradient of 0-3 N formic acid containing 10% ethanol.Thin layer chromatography. The fraction containing the inhibitory substanceobtained from the Dowex-l column was analysed by thin layer chromatographyusihg Merck precoated Kiesel Gel 60. After developing with n-butanol-aceticacid-water (5: 3 : 5) or ethanol-acetic acid-water (90: 5 : 5) as a mobile phase, itwas sprayed with ninhydrin solution for the detection of peptides.2Acta Medica Okayama, Vol. 32 [1978], Iss. 5, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss5/3GAP-Dehyd Inhibitor in Diabetes Mellitus 339RESULTS AND DISCUSSIONInhibitory effects of GAP-Dehyd in urine samples from a normal subjectand three diabetics are shown in Fig. 1. Inhibition of GAP-Dehyd was not100c.2.t:.c:c.5'tl>..c:c~ci. 50«Cl'0...CQl"a;Gl..010D.M. case 1y=57.0 log x-35.1•D.M. case 2.Y=46.0 log x-49.9~/No,m~200 500Volume of urine (pI!Fig. 1. Dose response of urinary inhibitory activity of GAP-Dehyd. Urine samples (10-500 pI) from a normal and three patientswith diabetes mellitus were added to the medium for assay of GAP·Dehyd activity and the GAP-Dehyd inhibitory activity was deter-mined as described in the text,observed with less than 200 ,ul of normal urine. However an appreciable inhibi-tion of GAP-Dehyd was observed with smaller amounts of diabetic urine (W-100 pI). The inhibition was proportional to the log dose of urine and paralleldose response curves were obtained. Many diabetic cases showed very greatinhibitory activity of GAP-Dehyd in the urine. The mean inhibitory activity of49 urine samples from 41 diabetic patients was 27.2±3.0% (mean±S. E. M.),while the inhibitory activity of urine from 17 normal volunteers was 9.0 ± 1.0 %.A significant difference of inhibitory activity was observed between normal anddiabetic urine (p<0.05). But, there were no significant correlation between theinhibitory activity and the degrees of severity (mild, moderate and severe) of thedisease. In order to clarify the differences of the urinary concentrarion of theinhibitory substance among the degrees of severity of the disease, further estima-tion of the other constitutions of urine, such as the concentration of creatinineand urea, will be required.The molecular weight of this inhibitor was determined by gel filtrationchromatography on Biogel P-4 and confirmed to be 100-300 daltons, which issimilar to that of the nonpeptide inhibitory substance described by Schwartz and3Terato et al.: An inhibitory substance of glyceraldehyde 3-phosphateProduced by The Berkeley Electronic Press, 1978340 K. TERATO et at.Turfus (2). One peak of inhibitory activity was found at the same effluent posi-tion with CoClz (Fig. 2).100o.~ 50>'';::;"co>-B:c:E£VitaminB12(MW1,300)Blue dextran(MW 2,000,000)1--------1I II II \I \ "[ I II {.! I f \ I \: V \ 1\I I J \I \ I Ir \_..... I \I \ i \I \ I \I ... '" \I II II 1J \i \, ..._----_..-- ....-2,0~(S'!!!.e-<D::J~.""1.0 aIVIIIo::J3IJII60,4020o t:-=-=-t..:t=:lo::.:-=--::-:'::-::::--:'::-=-~~J.,I..l,,---i.....L...:lL....- ---";L....-.__-.J 0.0oTube No.Fig. 2. Elution pattern of GAP-Dehyd inhibitory activity from Biogel P-4. Ninetypercent ethanol extract of urine (10 ml) from a diabetics (case 3) was dried in vacuoand dissolved in 2 ml of 0.05 M acetic acid. The solution was applied on a column ofBiogel P-4 (1.5x82 em) and eluted with the same solvent at 4°G. The effluent frac-tions (3 ml each) were collected at a rate of 10 ml/h, and the GAP-Dehyd inhibitoryactivity was determined using 100 fil of each effluent. The inhibitory activity wasobserved at tube No. 30-50 of the same effluent position with GoGlz.Furthermore, the physico-chemical property of this substance was similar tothat of the nonpeptide inhibitory substance which is adsorbed on anion exchangeresins, but different to that of the peptide inhibitor described by Bornstein et af.(1,4). By the addition of I m of Dowex-I (CI-) or DEAE-Sephadex to I ml ofurine which had been adjusted the pH to 6-9, the inhibitory activity disappearedcompletely from the supernatant, while, by the addition of Dowex-50 (H+) orcharcoal at pH 2-4, no decrease in the activity was observed.On the other hand, GAP-Dehyd was not substantially inhibited by theaddition of 0.2 ml of creatinine solution (l mg/ml) to the reaction mixture, in-dicating that the inhibitory substance of GAP-Dehyd in urine is not creatinine.And it is also different from urea, because this inhibitory substance of GAP-Dehyd had negative charge since it adsorbs to anion exchange resins.This substance was purified by anion exchange chromatography usingDowex-I (HCOO- type) from urinary samples exhibiting strong inhibitory activ-ity of GAP-Dehyd, which were obtained from a patient with diabetes mellitusand a patient of Werner's syndrome with glucose intolerance. The elution pat-terns from the Dowex-I (HCOO-) column by gradient elution of formic acid(0-3 N) are shown in Fig. 3. To avoid the non-specific adsorption of the inhibi-tory substance to the resin, ethanol was added to the elution buffer to make a4Acta Medica Okayama, Vol. 32 [1978], Iss. 5, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss5/3GAP-Dehyd Inhibitor in Diabetes Mellitus 3413Z2 :I:ooo1 J:3z2:I:ooo1. J:o700' 800FV·Fill.'400a)T.T..bIN.S.(Diabetes Mellitus).."\: \! \(Werner Synd. : iwith hyperglycemia) I ~ '": ~ (\: \ ! \; -_••J \!FIi-100 2.0~"0>-J:t..QD. c0« CD0 Nl. :i tQ aD. CD«. N0' ...:0 50 ~.o>- >-.t::.:t= "-.~ ~ ,/ \l;l ...> - ,i \~.~ ,,\~ 8- ".. \ FVI~ ,i \.,..... -0__-... ---..... 0o 0.00!<--~10~0=---::2,.:00=-"~~~~~50~0=-""-.6:-:0':"0~7~070--'--;8~00- ..o .?: 50 ~.O:~ °2 l~ -8 i\~ ~ ,:\,:i:c_~ 8- '",I .............o 0.0 -"'-'o 100100 2.0~Fig. 3. Elution diagrams of inhibitory activity of GAP-Dehyd from Dowex-l(HCeXT type) by gradient elution with 0-3 N formic acid.90% ethanol extracts of urine (500 ml) from a patient (T. T.) of Werner's syn-drome with glucose intolerance (al and urine (1,000 ml) from a patient (N. S.) withdiabetes (b) were applied on column of Dowex-l (HC(XT type) (1.5 x 25 cm or 3 x 45em) and were eluted at first with 200 ml (a) or 300 ml (b) of 10% ethanol and theneluted with 0-3 N formic acid gradient [600 ml (al or 1,000 ml (b)J containing ethanol(IO%).final concentration of 10%. The inhibitory substance in urine from both thepatients was similarly eluted at 0.3-0.7 N of formic acid.The fractions I-II in Fig. 3-a and fractions III-V in Fig. 3-b were lyophy-lized and then dissolved in 10 ml of 0.05 M acetic acid, respectively. One ml ofthe each solution was applied to Merck precoated Kiesel Gel 60 for preparation.After developing with two different solvent systems for 1-2 h, the inhibitorysubstance was eluted from 1cm silicagel bands with 0.05 M acetic acid. Theinhibitory activity of GAP-Dehyd was recovered from the thin layer plates at Rf0.4 and 0.9 (n-butanol-acetic acid-water (5: 3 : 5) and ethanol-acetic acid-water(90: 5: 5) solvent system) corresponding to a ninhydrin reacted spot. Therefore,the GAP-Dehyd inhibitor in urine was assumed to be a ninhydrin reactive sub-stance of low molecular weight, which has similar physico-chemical propertiesto the non-peptide inhibitory substance found in plasma by Schwartz and Turfus(2). But, at present, it is not certain whether the inhibitory substance of GAP-5Terato et al.: An inhibitory substance of glyceraldehyde 3-phosphateProduced by The Berkeley Electronic Press, 1978342 K. TERATO et al.Dehyd in urine is an identical compound to the inhibitory substance in plasma,and further purification of both the substances is required for identification andalso for biological studies on these substances.In this communication, we reported the presence of an inhibitory substanceof GAP-Dehyd in urine of diabetics, which may affect carbohydrate metabolism.The elucidation of the chemical and biological properties of this GAP-Dehydinhibitory substance will aid in solving some important problems on the abnormalcarbohydrate metabolism in diabetes mellitus.REFERENCES1. Bornstein,]., Taft, H. P., Armstrong, J. Me. D., Ng, F. M. and Gould, M. K.: The mecha-nism of the diabetogenic effects of pituitary growth hormone. Postgrad. Med. j. 49,(suppl), 219-242, 1973.2. Schwartz, P. L. and Turfus, 1. M.: Inhibition of glyceraldehyde 3-phosphate dehydroge-nase by plasma and serum ultrafiltrates due in part to a low molecular weight, non-peptide material. Metabolism 24, 569-572, 1975.3. Bornstein,]., Krahl, M. E., Marshall, L. B., Gould, M. K. and Armstrong, ]. Me. D. : Pitui-tary peptides with direct action on the metabolism of carbohydrates and fatty acids.Biochim. Biophys. Acta 156, 31-37, 1968.4. Zimmet, P., Ng, F. M., Bornstein, ]., Armstrong, J. Me. D. and Taft, H. P.: Insulin antag-onist of pituitary origin in plasma of normal and diabetic subjects. Br. Med.]. 1,203-204,1971.5. Bornstein,]., Armstrong, ]. Me. D. and Jones, M. D.: The effect of a growth hormonefraction on the activity of glyceraldehyde 3-phosphate dehydrogenase. Biochim. Biophys.Acta 156, 38-43, 1968.6Acta Medica Okayama, Vol. 32 [1978], Iss. 5, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss5/3

An inhibitory substance of glyceraldehyde 3-phosphate dehydrogenase in urine of diabetic patients

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