Biochemical and molecular medicine最新文献

{"title":"Differential Effects of Insulin-like Growth Factor-1 on Neonatal Canine Gene Expression","authors":"Bing-cheng Feng,&nbsp;Jixuan Li,&nbsp;Robert M. Kliegman","doi":"10.1006/bmme.1996.0081","DOIUrl":"10.1006/bmme.1996.0081","url":null,"abstract":"<div><p>To determine the effects of insulin-like growth factor-1 (IGF-1) and amylin on glucose homeostasis<em>in vivo</em>in newborn dogs, euglycemic hyper-IGF-1 clamps and hypoglycemic hyper-IGF-1 clamps were performed in newborn dogs. Northern blotting and radioimmunoassays were used to study the effects of the infused IGF-1 and/or hypoglycemia on the mRNA expression of the genes for phosphoenolpyruvate carboxykinase (PEPCK) and on the expression of the amylin gene in newborn dogs. Our results were that (1) Infused IGF-1 (plasma IGF-1 ≥1000 ng/ml) rapidly lowered the plasma glucose level, and 120 ± 38 mg glucose/pup was co-infused during a 105-min clamp to maintain the plasma glucose at the basal level. (2) The infused IGF-1 rapidly reduced the liver cytosolic mRNA for the PEPCK gene to an almost undetectable level. (3) Hyper-IGF-1 had no effect on mRNA level of the amylin gene in pancreas, 106.7 ± 14.2% vs 100.0 ± 5.9% (controls), or on plasma amylin concentration, 56.0 ± 5.7 pg/ml vs 52.1 ± 5.7 pg/ml (basal). (4) The amylin mRNA level, 127.8 ± 3.9% vs 100.0 ± 5.9% (controls) (<em>P</em>= 0.017), and the plasma amylin concentration, 132.3 ± 18.3 pg/ml vs 110.0 ± 10.8 pg/ml (controls) (<em>P</em>= 0.371), showed a parallel stimulation by hypoglycemia in the presence of hyper-IGF-1. We concluded that (1) IGF-1 acutely suppressed cytosolic PEPCK gene expression in liver of newborn dogs. (2) IGF-1 does not effect the expression of the pancreatic amylin gene. (3) Amylin may be involved in glucose homeostasis in newborn dogs and may play a role as a counterregulatory factor during the neonatal period. Unsuppressed amylin production may contribute to neonatal hyperglycemia.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 154-160"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0081","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19949842","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hexose Metabolism in Pancreatic Islets: Apparent Dissociation between the Secretory and Metabolic Effects ofD-Fructose","authors":"Abdullah Sener,&nbsp;Willy J. Malaisse","doi":"10.1006/bmme.1996.0085","DOIUrl":"10.1006/bmme.1996.0085","url":null,"abstract":"<div><p>In rat pancreatic islets,<span>D</span>-fructose causes a concentration-related shift to the left of the sigmoidal relationship between insulin release and<span>D</span>-glucose concentration. For instance, when<span>D</span>-fructose is tested at a 80 m<span>M</span>concentration, which is close to the threshold value for stimulation of insulin release by the ketohexose in the absence of<span>D</span>-glucose, a close-to-maximal secretory response is recorded in islets concomitantly exposed to as little as 6.0 to 8.3 m<span>M</span><span>D</span>-glucose. Under these conditions, however,<span>D</span>-fructose fails to affect the utilization of<span>D</span>-[5-³H]glucose, the oxidation of<span>D</span>-[U-¹⁴C]glucose, or its conversion to either¹⁴C-labeled acidic metabolites or amino acids. Under the same experimental conditions, the oxidation of<span>D</span>-[U-¹⁴C]fructose and its conversion to¹⁴C-labeled amino acids represent no more than 80–85% of the corresponding values found with 6 m<span>M</span><span>D</span>-[U-¹⁴C]glucose. Actually, the total output of¹⁴CO₂attributable to the oxidation of both<span>D</span>-[U-¹⁴C]glucose (6 m<span>M</span>) and<span>D</span>-[U-¹⁴C]fructose (80 m<span>M</span>) remains lower than that found in the sole presence of 8.3 m<span>M</span><span>D</span>-[U-¹⁴C]glucose, despite the much higher rate of insulin secretion found in the former compared to the latter situation. These findings suggest that the insulinotropic action of<span>D</span>-fructose cannot be fully accounted for by its capacity to act as a fuel in islet cells, as if it were to involve the generation of a second messenger distinct from those coupling factors currently implied in the process of nutrient-stimulated insulin release.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 182-186"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0085","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19949846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lung CuZn-Superoxide Dismutase and Catalase Gene Expression in Premature Rabbits Treated Intratracheally with Antioxidant-Surfactant Liposomes","authors":"Frans J. Walther ,&nbsp;Elba I. Mehta,&nbsp;James F. Padbury","doi":"10.1006/bmme.1996.0083","DOIUrl":"10.1006/bmme.1996.0083","url":null,"abstract":"<div><p>Tolerance to hyperoxia usually depends on an increase in lung antioxidant enzyme activity. Ant- ioxidant-surfactant liposomes, encapsulating the antioxidant enzymes CuZn-superoxide dismutase (CuZnSOD) and catalase in synthetic surfactant lipids, increase lung antioxidant activity following intratracheal instillation in premature and term rabbits. We investigated whether the exogenous antioxidant enzymes encapsulated in these liposomes inhibit the endogenous antioxidant enzyme synthesis in the premature rabbit lung. Premature rabbits, delivered at 28 days of gestation, were treated intratracheally with antioxidant-surfactant liposomes, surfactant liposomes without antioxidant enzymes, or air placebo at birth and exposed to hyperoxia for 24 h. A comparison group was killed after breathing room air at birth. The right lungs of the pups were assayed for CuZnSOD and catalase activities and DNA content, the left lungs of the same pups were used to quantitate the concentrations of CuZnSOD and catalase mRNA using cRNA probes. Lung CuZnSOD and catalase mRNA quantities increased during exposure to hyperoxia, but were not affected by exogenous antioxidant enzymes. These data suggest that intratracheal instillation of CuZnSOD and catalase does not down-regulate mRNA transcription of these antioxidant enzymes in the premature rabbit lung.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 169-173"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0083","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19949844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cathepsin K: Isolation and Characterization of the Murine cDNA and Genomic Sequence, the Homologue of the Human Pycnodysostosis Gene","authors":"Bruce D. Gelb ,&nbsp;Konstadinos Moissoglu ,&nbsp;Jian Zhang ,&nbsp;John A. Martignetti ,&nbsp;Dieter Brömme ,&nbsp;Robert J. Desnick","doi":"10.1006/bmme.1996.0088","DOIUrl":"10.1006/bmme.1996.0088","url":null,"abstract":"<div><p>Cathepsin K(EC 3.4.22.38) is a lysosomal cysteine protease that is strongly implicated in bone resorption. The human cathepsin K gene is highly expressed in osteoclasts and gene mutations cause pycnodysostosis, an autosomal recessive skeletal dysplasia. To investigate the evolutionary relatedness of cathepsin K across species, the mouse cathepsin K gene was isolated. A mouse heart cDNA clone, pMCatKl, contained the 3′ untranslated region, mature enzyme coding sequence, and most of the propeptide. The remainder of the gene was amplified from mouse melanocyte RNA using 5′ rapid amplification of cDNA ends. The gene contained a 990-bp open reading frame, predicting a 329-amino-acid prepropolypeptide. The structure of the protein included a 15-amino-acid presignal, a 99-amino-acid proregion, and a 215-amino-acid mature enzyme. Two potential N-glycosylation sites were identified, one in the proregion and one in the mature enzyme. The 5′ untranslated region was 135 bp. The 3′ untranslated region was 470 bp including a 9-bp poly(A) tract and contained two polyadenylation signals. The mouse cathepsin K nucleotide and amino acid sequences were highly conserved with the human, rabbit, and chicken homologues across the proregion and mature enzyme. The mouse cathepsin K gene was isolated from an V129 genomic library, and characterization of its genomic structure and intron sizes revealed exons with the initiation ATG in exon 2 and termination TGA in exon 8, a genomic organization that was highly conserved with its human homologue. The availability of the mouse cathepsin K cDNA and genomic sequences will facilitate generation of a mouse model of cathepsin K deficiency by gene targeting.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 200-206"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0088","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19948040","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Influence of Dietary Folate and Methionine on the Metabolic Disposition of Endotoxic Homocysteine","authors":"M.D. Lucock ,&nbsp;I.G. Daskalakis ,&nbsp;J. Wild ,&nbsp;A. Anderson ,&nbsp;C.J. Schorah ,&nbsp;M.E.J. Lean ,&nbsp;M.I. Levene","doi":"10.1006/bmme.1996.0074","DOIUrl":"10.1006/bmme.1996.0074","url":null,"abstract":"<div><p>We have investigated the disposition of potentially endotoxic homocysteine (Hcy) and its transsulfuration metabolite cysteine (Cys) in 98 individuals (age range 20–66 years). Our study reports on the relationship between Hcy and two important dietary factors likely to influence plasma levels of this thiol: dietary folate and dietary methionine. χ²analysis shows a low frequency of elevated plasma Hcy at high folate intake. This frequency for Hcy &gt;10 μmol/liter with a folate intake &gt;350 μg/day is significant (<em>P</em>&lt; 0.02). The data reflect a tendency for elevated Hcy values to be associated with low dietary folate, although many subjects with a low dietary folate also had a low plasma Hcy. Intake of dietary methionine was found to be significantly higher in males than in females (<em>P</em>&lt; .0001). This may account for the looser relationship between Hcy and its transsulfuration product, Cys, in females (<em>R</em>²= 0.30) compared to males (<em>R</em>²= 0.73), since conversion of methionine to SAM in males would activate cystathionine β synthase and commit excess Hcy to transsulfuration. The generally lower methionine intake of females means that more Hcy is utilized in the remethylation cycle in which methionine is produced from the<em>de novo</em>methyl group of 5-methyltetrahydrofolate or from the preformed methyl group of betaine. Clearly a Hcy moiety locked up in remethylation would be further removed from Cys, the end product of transsulfuration. An increasing number of studies are clarifying the relationship between Hcy, folate, and other B vitamins. However, less attention seems to be given to the influence of dietary methionine on the disposition of Hcy. The present study supports biochemical theory and indicates that more focus should be given to the effect of dietary methionine on Hcy. These findings have particular significance since even moderate increases in plasma Hcy are associated with a toxic vascular effect. Consequently the relationship between dietary folate and Hcy levels should be a factor in evaluating recommended dietary allowances for this vitamin. The simplicity of our dietary folate questionnaire also raises the possibility of a screening test in which individuals can ascertain whether their folate intake is adequate to reduce Hcy levels to a benign value.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 104-111"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0074","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19947592","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) in Rat Testis Nuclei Is Bound to Chromatin","authors":"Cristiana Godeas ,&nbsp;Federica Tramer ,&nbsp;Fulvio Micali ,&nbsp;Antonella Roveri ,&nbsp;Matilde Maiorino ,&nbsp;Carla Nisii ,&nbsp;Gabriella Sandri ,&nbsp;Enrico Panfili","doi":"10.1006/bmme.1996.0076","DOIUrl":"10.1006/bmme.1996.0076","url":null,"abstract":"<div><p>In rat testis nuclei the activity of the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx, EC 1.11.1.12) is much higher than in other tissues and subcellular compartments, with the sole exception of mitochondria. In nuclei, the bound enzyme is solubilized by DNase I treatment, thus suggesting a binding to chromatin. Treatment with ionic strength releases about 70% of bound PHGPx, suggesting that electrostatic bonds are involved. Immunogold electron microscopy indicates the association of PHGPx with chromatin structures in isolated nuclei. A possible interpretation of these data is a PHGPx protective role against DNA peroxidative damage. Furthermore, in agreement with kinetic and structural information, PHGPx-chromatin binding could suggest an hypothetical thiol oxidase activity toward specific thiol bearing proteins which could substitute for GSH as alternative donor substrates. Such activity could give to the enzyme a new important function which is not only protective but also has a specific regulatory function in chromatin condensation.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 118-124"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0076","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19947594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toxicity of Cationic Lipid–Ribozyme Complexes in Human Prostate Tumor Cells Can Mimic Ribozyme Activity","authors":"Stephen J. Freedland,&nbsp;Robert W. Malone,&nbsp;Holger M. Borchers,&nbsp;Zhanna Zadourian,&nbsp;Jill G. Malone,&nbsp;Michael J. Bennett,&nbsp;Michael H. Nantz,&nbsp;Juan-Hua Li,&nbsp;Paul H. Gumerlock,&nbsp;Kent L. Erickson","doi":"10.1006/bmme.1996.0080","DOIUrl":"10.1006/bmme.1996.0080","url":null,"abstract":"<div><p>Prostate tumor cell lines have been shown to both produce interleukin-6 (IL-6) and express the IL-6 receptor, suggesting a potential autocrine growth regulatory role for IL-6. We explored the role of IL-6 in the proliferation of the human prostatic carcinoma cell line, DU145, using ribozymes to inhibit IL-6 expression. Hammerhead-type ribozymes targeted against IL-6 mRNA sequences were prepared, and<em>in vitro</em>analyses were used to demonstrate that these molecules catalyzed the cleavage of IL-6 mRNA poly- nucleotide fragments. To test<em>in situ</em>activity, these ribozymes were transfected into DU145 cells using cationic transfection lipids, cytofectins. Treatment of cultured cells with ribozyme/cationic lipid complexes resulted in a reduction of IL-6 protein levels in the supernatant and reduced numbers of DU145 cells 48 h after treatment. However, similar results were also seen following treatment with control RNA/lipid complexes. This reduction in IL-6 levels and cell numbers was a function of the RNA/lipid complexes and was not seen with either lipid or RNA alone. Therefore, the reductions in IL-6 levels and cell numbers observed were not due to ribozyme-mediated cleavage of IL-6 mRNA, but rather reflected a dose-dependent, nonspecific toxic effect of the treatment with ribozyme/cytofectin complexes. This effect can resemble functional ribozyme activity, complicating analysis of the activity of synthetic ribozymes after transfection into cultured cells.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 144-153"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0080","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19949841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Development of Insulin Resistance with High Fat Feeding in Rats Does Not Involve Either Decreased Insulin Receptor Tyrosine Kinase Activity or Membrane Glycoprotein PC-1","authors":"Begüm Özel ,&nbsp;Jack F. Youngren ,&nbsp;Jason K. Kim ,&nbsp;Ira D. Goldfine ,&nbsp;Chin K. Sung ,&nbsp;Jang H. Youn","doi":"10.1006/bmme.1996.0084","DOIUrl":"10.1006/bmme.1996.0084","url":null,"abstract":"<div><p>Recent studies have suggested that the insulin receptor tyrosine kinase inhibitor, membrane glycoprotein PC-1, may play a role in certain insulin resistant states. In the present study, we examined whether either insulin receptor function or PC-1 activity was altered during the development of insulin resistance that occurs with high fat feeding in normal rats. Over the course of 14 days of high fat feeding, both maximal and submaximal (physiological) insulin-stimulated skeletal muscle glucose uptake decreased gradually; after 14 days of high fat feeding, submaximal and maximal insulin-stimulated glucose uptake decreased by ∼40 and ∼50%, respectively. In contrast, in the same muscles (tibialis anterior) of these animals, neither insulin receptor content nor insulin-stimulated insulin receptor autophosphorylation was altered after 14 days of high fat feeding. PC-1 has both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) enzyme activities. These enzyme activities showed no changes during the course of 14 days of high fat feeding. Individual data revealed that there was no significant correlation between insulin-stimulated glucose uptake and alkaline phosphodiesterase or nucleotide pyrophosphatase activity (<em>P</em>&gt; 0.05). Together, these data indicate that neither defects in insulin receptor function nor elevated PC-1 activities are involved in the development of insulin resistance in rats with high fat feeding, and the insulin resistance induced with high fat feeding is likely due to postreceptor defects in skeletal muscle.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 174-181"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0084","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19949845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dideoxyfingerprinting (ddF) Analysis of the Type X Collagen Gene (COL10A1) and Identification of a Novel Mutation (S671P) in a Kindred with Schmid Metaphyseal Chondrodysplasia","authors":"Constantine A. Stratakis ,&nbsp;Zsolt Orban ,&nbsp;A.Lee Burns ,&nbsp;Alessandra Vottero ,&nbsp;Constantine S. Mitsiades ,&nbsp;Stephen J. Marx ,&nbsp;Val Abbassi ,&nbsp;George P. Chrousos","doi":"10.1006/bmme.1996.0075","DOIUrl":"10.1006/bmme.1996.0075","url":null,"abstract":"<div><p>Schmid metaphyseal chondrodysplasia (SMCD; MIM 156500) is an autosomal dominant disorder of the skeleton that is manifested in early childhood by short stature,<em>coxa vara,</em>and a waddling gait. Patients with SMCD have mutations in the gene that codes for the α-1 chain of collagen X (COL10A1); however, mutation analysis of this gene is hampered by its size. We studied a family with SMCD: the mother, a 36-year-old woman with a height of 149 cm, had mild bilateral<em>coxa vara.</em>Her two sons presented with short stature, bowed legs, and<em>coxa vara</em>in early childhood. DNA was extracted from peripheral lymphocytes from the three patients and subjected to PCR amplification by COL10A1 gene-specific primers. In addition to single-strand conformational polymorphism (SSCP) analysis of the COL10A1 gene, we used a novel method, dideoxy fingerprinting (ddF). The genetic defect in this family was found to be a previously unreported missense mutation (T-to-C transition) at nucleotide 2011. This change resulted in a Ser-to-Pro substitution at position 671 of the carboxy-terminus of the COL10A1 protein. In addition, the two boys, but not the mother, were found to carry a trinucleotide (CCC) deletion at position 2048 of the 3′ untranslated region, a polymorphism of the COL10A1 gene. We conclude that ddF can be used in the analysis of the COL10A1 gene along with SSCP. The S671P substitution is novel, but located in the same region with the other reported COL10A1 mutations, confirming type X collagen as the locus for this disease.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 112-117"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0075","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19947593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A New Type of Major Aminopeptidase in Bovine Brain","authors":"Jungo Yanagisawa ,&nbsp;Michio Tsuda ,&nbsp;Tomoichi Ohkubo ,&nbsp;Mineyoshi Hiyoshi ,&nbsp;Hiroshi Kamiguchi ,&nbsp;Hideo Tsukamoto ,&nbsp;Masaichi Yamamura ,&nbsp;Tomoji Kocha ,&nbsp;Takaaki Aoyagi","doi":"10.1006/bmme.1996.0082","DOIUrl":"10.1006/bmme.1996.0082","url":null,"abstract":"<div><p>A new type of major aminopeptidase was purified from bovine brain by ammonium sulfate fractionation and TMAE-fractogel (anion exchange), arginine–Sepharose 4B, Sephadex G-150, and Sephadex G-100 column chromatography. The purified enzyme showed a maximum activity at pH 7.2, and its molecular size was estimated to be 98,000 by gel filtration and 104,000 by SDS–PAGE with or without 2-mercaptoethanol. Further properties were activation by thiol reagents; inhibition by EDTA, puromycin, bestatin, amastatin, actinonin, leuhistin and probestin; and very low concentrations of Cu²⁺, Cd²⁺, Pb²⁺, Al³⁺, Fe³⁺, and Zn²⁺inhibited activity. The enzyme hydrolyzed several amino acyl-7-amido-4-methylcoumalin derivatives (amino acid-MCA). The order of MCA-substrate specificity expressed as kcat/<em>K</em>_<em>m</em>is Lys-MCA &gt; Arg-MCA &gt; Leu-MCA &gt; Met-MCA &gt; Phe-MCA &gt; Tyr-MCA &gt; Ala-MCA &gt;&gt; Gly-MCA, Pro-MCA, Ser-MCA, Asn-MCA. Immunoreactivity of the antibody against the purified aminopeptidase was observed in human brain and most rat tissues examined including brain, liver, kidney, lung, heart, and skeletal muscle at the same molecular size as in bovine brain aminopeptidase. Most of the Lys-, Leu-, Met-, and Phe-MCA degrading activity in crude bovine and human brain extracts was absorbed by the aminopeptidase IgG, suggesting that this aminopeptidase is a major enzyme, sharing at least Lys-, Leu-, Met-, and Phe-MCA degrading aminopeptidase activities in the brains.</p></div>","PeriodicalId":8837,"journal":{"name":"Biochemical and molecular medicine","volume":"59 2","pages":"Pages 161-168"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/bmme.1996.0082","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19949843","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}