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Variants of Phosphohexose Isomerase in Gastrointestinal And

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[CANCER RESEARCH 48. 2998-3001, June, 1988] Variants of Phosphohexose Isomerase in Gastrointestinal and Mammary Carcinoma: Isoelectric Focusing Patterns of Normal and Tumor Tissues Derived from Surgical Specimens of the Same Patient1 Matthias Baumann, Damián Jezussek, Ralf-Torsten Richter, and Karl Brand2 Institute of Biochemistry, Medical Faculty, University of Erlangen-Nuremberg, Federal Republic of Germany ABSTRACT H2PO4 H2O, 5; MgSO4 7H2O,0.77; Krebs saline buffer: H2O, 1:4; pH 7.5). The occurrence of charge variants of phosphohexose isómeras? was Then the homogenate was sonicated by 6 pulses of 10 s each at 50 elucidated in cancerous and, for comparison, in noncancerous tissues W with cooling intervals of 1 min to make sure that the temperature of the preparation remained below 10'C. Subsequently, the extract was obtained from the same organ. Surgical specimens from 35 patients with gastrointestinal and mammary carcinoma were studied by means of the rehomogenized and centrifuged at 12,000 x g for 10 min. The super isoelectric focusing (IEF) technique. Differences in the IEF pattern could natant was dialyzed against 1% glycine buffer, pH 7.5, overnight at 4'C. LKB Multiplier 2117, LKB Power Supply Unit 2103, and Servalyt be demonstrated in 90% of the patients with gastric cancer. Correspond ing values for the patients with colorectal and mammary carcinoma were Precotes, pH 3-10, were used for the isoelectric focusing experiments. 50 and 73%, respectively. Almost all normal specimens proved to be A Julabo Paratherm FT20p electronic thermostat served to cool the plate to 4'C during the run. monomorphic, revealing only the major basic band with a pi of 9.1. In most of the tumors, additional bands of pi 8.9 and 8.6 were found. This The dialyzed probes were diluted with 2% LKB ampholine, pH 3- "typical" IEF pattern in tumor tissues was observed in 60% of gastric 10, achieving a final PHI activity of 1000-3000 »¿unitsina sample and 73% of mammary tumors, but in only 36% of colorectal tumors. The volume of 20 ¡A.Aconstant power of 4.0 W was preset and the Precote results obtained suggest that the IEF pattern of phosphohexose isomerase was prefocused until the voltage reached 500 V. Then the probes were in tumor tissue might be a useful tool for tumor diagnosis. applied and a voltage limit of 2000 V was chosen. When this voltage was reached the run was carried out for a further 30 min, so that the whole procedure took 3-4 h. INTRODUCTION Subsequently the precote was specifically stained for PHI, according to the method described by Carter and Parr (25), using phenazine The analysis of electrophoretic variants of the glycolytic methosulfate and the tetrazolium salt 3-(4,5-dimethyl-2-thiazolyl)-2,5- enzyme PHI3 is a widespread method in human genetics and diphenyl-2H-tetrazolium bromide. For each run, the protein standard internal medicine (1-7). Furthermore, serum PHI activity is a Test Mix 9, supplied by Serva, was used to determine the isoelectric well established tumor marker in oncology (8-19). The molec points (pi) of the PHI bands. This standard was visualized by Coomassie ular and kinetic properties of human PHI were investigated by staining. PHI activity was assayed spectrophometrically according to using hemolysates and muscle extracts as enzyme source (20- Tilley et al. (2). 22). There are relatively few reports dealing with the quantita tive and, in particular, qualitative comparison of PHI in normal and tumor tissues derived from surgical specimens of the same RESULTS patient (23, 24). Thirty-five patients with malignant tumors were included in The purpose of the present study was to evaluate the occur rence of PHI variants, comparing authentic normal and tumor this comparative study, 10 with gastric, 14 with colorectal, and tissues derived from the same organ immediately after surgical 11 with mammary carcinoma. By IEF, three charge variants of removal. Tissue specimens from 35 patients with gastric, colo PHI could be demonstrated in the tissues studied; a major basic rectal, and mammary carcinoma were studied by means of the band A with an isoelectric point of 9.1, and two more acidic IEF technique. bands B and C, with a pi of 8.9 and 8.6, respectively. In almost all normal specimens only the major band A could be found. In most tumor tissues additional bands could be detected. On MATERIALS AND METHODS the basis of this observation a "typical" IEF pattern of PHI, which is illustrated in Fig. 1, was defined as follows: normal Normal (noncancerous) and tumor tissue probes from the same organ tissue containing only band A and tumor specimen revealing of the patient were obtained immediately after surgical removal and two additional bands B and C. histopathological examination of each specimen. Only solid, surgically The IEF patterns of PHI found in normal and tumor tissue and histologically homogeneous tumor tissue, and closest noninvolved, histologically normal tissue as control were used. Blood and coagula specimens derived from the same organ of the patients are given in Tables 1-3. Table 1 shows that for gastric cancer PHI band were removed very thoroughly. All further steps were carried out at 4'C. Normal and carcinoma specimens were prepared identically. An A could be detected in all normal and tumor probes, whereas aliquot of 400 mg of each specimen was minced and homogenized in a band B is found in all tumor samples but only in 20% of the 10-fold volume of hypotonie Krebs saline buffer (in HIMconcentrations: normal specimens. Band C appeared in 8 of 10 malignant NaCl, 136; KC1, 4.7; CaCl2 2H2O, 1.29; K2HPO4 3H2O, 1.18; Na- probes but only in 1 of 10 benign tissues. In Table 2 the results obtained from the patients with colo Received 6/16/87; revised 12/10/87; accepted 2/23/88. The costs of publication of this article were defrayed in part by the payment rectal carcinoma are listed. In all 14 cases the normal tissue of page charges. This article must therefore be hereby marked advertisement in contained only band A. With respect to the tumor specimens, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by the Marohn-Stiftung Grant Nr. Br/85. additional bands B and C were found, however, in only SOand 2To whom requests for reprints should be addressed, at Institute of Biochem 36% of the patients. In one instance only band B and C, but istry, Medical Faculty, Fahrstraße17, D-8520 Erlangen, Federal Republic of not band A (pi 9.1) could be detected. Germany.. 3The abbreviations used are: PHI, phosphohexose isomerase; IEF, isoelectric Table 3 illustrates that all normal breast tissues revealed band focusing. A exclusively. In 73% of the malignant breast tissues band B 2998 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. VARIANTS OF PHI IN CANCER GASTRIC CARCINOMA COLORECTAL CARCINOMA MAMMARY CARCINOMA •• NORMAL TUMOR NORMAL TUMOR NORMAL TUMOR Fig. 1. Isoelectric focusing patterns of phosphohexose isomerase in normal (noncancerous) and tumor tissues from patients with gastrointestinal and mammary carcinoma. Aliquots of extracts from normal and tumor tissues were subjected to isoelectric focusing on Servalyt Precotes (3-10), as described in "Materials and Methods." Band A, pi 9.1; Band B, pi 8.9; Band C, pi 8.6. Table 1 Isoelectricfocusing patterns of phosphohexose isomerase in normal and Table 3 Isoelectricfocusing patterns of phosphohexose isomerase in normal and tumor tissues from patients with gastric carcinoma tumor tissues from patients with mammary carcinoma TumorBand B+*--A° Band B BandC BandA Band Patient1234S678910n%NormalBandB+»+A* Band C BandC+ A Band B Band Patient1234567891011n%Normal ++ + +— + —+ ++ + + ++ — — + ++ +— + + —+ — — + ++ +— + + ++ — — + —4. +— + + ++ - - + —+ —— + + —+ — — + —+ +— + + ++ — — + —+ —- + -t- ++ — — + —+10 +++ + —+ — — + +1 + ++ — — + +11 + 2100 810 10 10 8100 0 0 11 20TumorBand 100 100 80 " Band A, pi 9.1; Band B, pi 8.9, Band C, pi 8.6. 0 0 100 73BandC++—++—++—++873 * +, presence of band; —,absenceof band. " BandA, pi 9.1; Band B,pi 8.9; BandC, pi 8.6. +, presenceof band; -, absenceof band. Table 4 Comparison of isoelectricfocusing patterns of phosphohexose isomerase in normal and tumor tissues obtained by surgical removal from the same patient The typical IEF pattern of PHI is defined: in the noncancerous tissue only band A, but bands A, B, and C in the tumor tissue. Table 2 Isoelectricfocusing patterns of phosphohexose isomerase in normal and tumor tissues from patients with colorectal carcinoma of patients with differentIEF pat of patients with terns in normal typicalIEF pat TumorGastric and tumortissues90 tern60 Patient1234S6791011121314m%NormalBandB+»-1-A* Band C BandC+ A Band B Band +— •+ carcinoma —+ +— -1- + Colorectalcarcinoma 14 SO 367351 —+ —— + — MammarycarcinomaAlln10 1135% 7369% —++ —— + — —— + — —+* —- + — -! + - I+ -- + + and C could be detected in addition to band A. These data are -+ -—+ - summarized in Table 4. —+ —-+ + In 90% of the patients with gastric carcinoma a difference in —+ +—+ + the IEF pattern of PHI between normal and tumor tissue —+14 +- — + -0 + - derived from the same patient could be demonstrated. Corre sponding values for colorectal and mammary carcinoma were 0100 50 13 7 50 and 73%, respectively. The "typical" IEF pattern of PHI as 0TumorBand 93 50 36 defined above (Fig. 1) occurred in 60 and 73% of all cases with * Band A, pi 9.1; Band B, pi 8.9; Band C, pi 8.6.
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    Scaffoldless engineered enzyme assembly for enhanced methanol utilization J. Vincent Pricea, Long Chena, W. Brian Whitakera,b, Eleftherios Papoutsakisa,b, and Wilfred Chena,1 aDepartment of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716; and bThe Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711 Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved September 27, 2016 (received for review February 4, 2016) Methanol is an important feedstock derived from natural gas and organisms requires electrons in the form of NADH and culture can be chemically converted into commodity and specialty chem- conditions largely microaerobic or anaerobic (7, 10). Thus, or- icals at high pressure and temperature. Although biological ganisms that can grow anaerobically or microaerobically and NAD- conversion of methanol can proceed at ambient conditions, there dependent Mdhs are essential for effective conversion of methanol + is a dearth of engineered microorganisms that use methanol to to desirable metabolites (7). Although NAD(P) -dependent Mdhs produce metabolites. In nature, methanol dehydrogenase (Mdh), are the best candidates for engineering synthetic methylotrophs which converts methanol to formaldehyde, highly favors the reverse like Escherichia coli, these enzymes have poor predicted thermo- reaction. Thus, efficient coupling with the irreversible sequestration dynamic properties and are thus dependent on maintaining low of formaldehyde by 3-hexulose-6-phosphate synthase (Hps)