Biomedical subjects

W Kreisel

Publications and source records attributed to W Kreisel.

At least 37 records · Page 2Linked to original sources

PubMed1996

Partial characterization of a new nucleotide binding glycoprotein of hepatocyte plasma membrane.

Hepatocyte plasma membranes contain a glycosylated 230-kDa Ca(2+) -dependent, Mg(2+)-stimulated ATPase (pgp230), which consists of two subunits, one of 120 kDa and the other of 110 kDa. pgp230 can be enriched by the use of affinity chromatography on Concanavalin A-Sepharose, wheat germ lectin-Sepharose, and 5'-AMP-Sepharose. It has a high-affinity Ca2+ binding site. In the presence of Ca2+, it forms a phosphorylated intermediate by autocatalytic transfer of the terminal phosphate residue from ATP. Maximal Ca(2+)-dependent autophosphorylation is observed at pH 5-6. Photoaffinity labeling using 8-azido-[alpha-32P]ATP or [y-32P]ATP confirms the presence of ATP binding sites. Incubation with [alpha-32P]ATP leads to a rapid but transient labeling of pgp230. Various nucleotides, nucleotide receptor agonists, or antagonists inhibit Ca(2+)-dependent phosphorylation by [y-32P]ATP. The concentrations of half-maximal inhibition range from 10(-7) M to 10(-3) M. The rank order of inhibitory potency is: ATP > alpha,beta-methylene-ATP > CTP = TTP > y-4-amino-phenyl-ATP = 2-methyl-thio-ATP > UTP = GTP > GDP = ADP = beta,y-methylene-ATP = beta, y-methylene-TTP = beta,y-methylene-GTP = adenosine-5'-O-2-thiodiphosphate = CMP = AMP > adenosine > cytidine > guanosine = suramin > Reactive blue 2 > iso-butyl-methyl-xanthine > thymidine > uridine. These data suggest a nucleotide binding capacity of this new hepatocyte membrane glycoprotein. Further investigations should be carried out to reveal its biological function.

Adenosine Triphosphate↗

PubMed1996

Renal tubular damage: an extraintestinal manifestation of chronic inflammatory bowel disease.

OBJECTIVE: To investigate whether treatment of inflammatory bowel disease (IBD) with 5-aminosalicylate or sulphasalazine in IBD may induce renal tubular damage. DESIGN AND METHODS: The urinary enzymes beta-N-acetyl-D-glucosaminidase ( beta-NAG), dipeptidylpeptidase 4 (DPP4) and alanine aminopeptidase (AAP) were measured as markers of renal tubular damage in 104 consecutive patients with Crohn's disease and in 43 consecutive patients with ulcerative colitis (all with normal serum creatinine values). Control values were gained from 65 healthy persons. RESULTS: The normal values (mean +/- SD) for the urinary enzymes investigated (U/g creatinine in the urine) were: DPP4 4.5 +/- 2.2, beta-NAG 1.6 +/- 1.4, AAP 11.4 +/- 6.5. In 28% of the patients with ulcerative colitis elevated beta-NAG levels of more than the mean + 2 x SD were measured. This pathological enzymuria was nearly exclusively found in patients with active disease (CAI > 6): DPP4 15.6 +/- 25.3, beta-NAG 8.3 +/- 10.1, AAP 24.7 +/- 50.1 (all three enzymes were significantly elevated). The highest values were measured in patients with active ulcerative colitis before start of therapy. Nineteen per cent of the patients with Crohn's disease had elevated beta-NAG levels of more than the mean + 2 x SD. There was no significant difference in enzymuria between patients with active (CDAI > 150) and patients with inactive Crohn's disease (CDAI < or = 150). DPP4 and AAP were normal in both groups. A correlation between the enzymuria and the cumulative doses of 5-aminosalicylic acid, sulphasalazine or prednisolone could not be found. The courses of enzymuria in three patients who presented with the first severe manifestation of IBD are described. They were treated with either corticosteroids and 5-aminosalicylic acid or corticosteroids and sulphasalazine. Before onset of therapy, very high urine enzyme values were measured. They almost normalized in the course of successful medical therapy despite increasing cumulative doses of 5-aminosalicylic acid or sulphasalazine. CONCLUSIONS: Renal tubular damage can frequently be observed in IBD. Our results suggest that this is an extraintestinal manifestation of the disease and not a toxic side-effect of anti-inflammatory therapy using 5-aminosalicylic acid or sulphasalazine.

Adolescent↗

PubMed1995

[Collagenous sprue].

HISTORY AND FINDINGS: A 45-year-old man with type I diabetes mellitus was admitted to hospital because of colicky abdominal pain and 5-6 watery stools daily. Upper gastrointestinal endoscopy showed nearly total atrophy of the villi in the duodenum and jejunum suggesting coeliac disease. However, gluten-free diet for 2 weeks brought no improvement. Another examination of the biopsy 6 weeks after the first endoscopy revealed extensive collagen deposition in the lamina propria of the small intestine, giving the diagnosis of collagenous sprue. TREATMENT AND COURSE: Parenteral nutrition, lactulose, cisapride, cholestyramine, doxycycline, paromomycin, vancomycin and octreotide failed to affect the loss of fluid from the gut which 12 weeks after admission had increased to 221 daily. However, it was stopped after prednisolone was administered (100mg daily). 7 months after starting the steroid treatment the collagen layer had disappeared and the villous atrophy had partially regressed. Over the next 6 months the prednisolone dosage was decreased to 10 mg daily. Shortly thereafter a perimembranous glomerulonephritis occurred, with proteinuria (up to 60 g/d) and oedema. It regressed to 6 g/d when the steroid dose was increased and cyclosporin, 0.5 g/d, had been added. On maintenance dosage of cyclosporin the histological and clinical remission of the collagenous sprue has now lasted for over 2 years. CONCLUSIONS: This case suggests that steroid administration is an effective treatment of collagenous sprue. The presence of diabetes and other immune-related diseases in this case also suggests that an immunological mechanism may play a causative role in collagenous sprue.

Anti-Inflammatory Agents↗

PubMed1995

International program on the health effects of the Chernobyl accident.

The International Program on the Health Effects of the Chernobyl Accident (IPHECA) was established by the World Health Organization (WHO) in 1991. Currently, the technical part of IPHECA consists of five projects addressing the following areas of priority health problems or needs: thyroid, hematology, brain damage in utero, epidemiological registry and oral health. Important findings are: 1) a significant increase of thyroid cancer in children in Belarus and Ukraine since 1989, and in Russia since 1992 though not so pronounced. A relationship between detected thyroid cancers and radiation exposure is yet to be established, 2) no increase yet in the incidence of hemoblastoses in the three States, 3) no relationship established between mental retardation and radiation exposure in utero in 4,500 children investigated. The importance of dosimetry and biological indicators of radiation damage has been recognized by IPHECA. Several methods of biological and physical dosimetry are being employed using instrumentation provided by IPHECA. Some preliminary results indicate: 1) unstable aberrations can indicate an integral exposure but it is heavily biased to recent exposures, 2) when comparing healthy persons and patients with hematological diseases in contaminated areas, there is a higher ratio of total aberrations compared to their background and that the level of stable is lower than unstable aberrations, and 3) by applying electron spin resonance (ESR) it has been shown that the individual distribution of doses approaches a log-normal one, especially for adults, and that a peak shift towards higher doses is noticeable for children.

Adult↗

PubMed1994

A new type of Ca(2+)-dependent, Mg(2+)-stimulated ATPase of rat liver plasma membrane.

Incubation of a glycoprotein fraction obtained from rat liver plasma membrane which has been previously well characterized using [gamma-32P]ATP results in the phosphorylation of a 230-kDa glycoprotein (pgp230). It is composed of a 120-kDa subunit (pgp120) and a 110-kDa subunit (pgp110) linked by interchain disulfide bonds. Peptide maps of pgp120 and pgp110 suggest extensive similarity in their polypeptide chains. Glycan analysis reveals between four and six hybrid-type oligosaccharide chains for both phosphoproteins. Immunoblotting using monoclonal antibodies and endoglycosidase digestion exclude an identity of pgp120 or pgp110 with the hepatocyte plasma membrane glycoproteins dipeptidylpeptidase IV or the taurocholate transport protein, which co-purify and co-migrate in SDS/PAGE. Protein phosphorylation is Ca(2+)-dependent (K0.5(Ca2+) = 0.35 microM, in the absence of Mg2+). In the presence of Mg2+, the glycoprotein undergoes rapid cycles of phosphorylation and dephosphorylation, resulting in ATPase activity. Analysis of phosphorylated amino acids identifies phosphothreonine as the major one. Photoaffinity labeling with 8-azido-[alpha-32P]ATP demonstrates the presence of one or more ATP binding site(s). Preincubation of pgp230 with various purine or pyrimidine nucleotides (ATP, UTP, TTP, ADP, GDP, AMP, CMP) or known P2-purinoceptor agonists or antagonists (adenosine 5'-[alpha,beta-methylene]triphosphate, 2-methyl-thio-adenosine 5'-triphosphate, suramin) inhibits its phosphorylation by [gamma-32P]ATP. The biological function of pgp230 is unknown at present. Several findings of the present study are compatible with the idea that pgp230 may be involved in a P2-purinoceptor function of the hepatocyte. Following this concept, a mechanism is discussed where a cytosolically exposed high-affinity Ca(2+)-binding site of pgp230 would allow for receptor feedback control, via phosphorylation and dephosphorylation, by sensing changes in cytosolic Ca2+ concentration.

Adenosine Triphosphate↗

PubMed1994

Immuno-gold electron microscopical detection of heat shock protein 60 (hsp60) in mitochondria of rat hepatocytes and myocardiocytes.

We characterize the specificity of a polyclonal antibody against heat shock protein 60 (hsp60) and present an application for ultrastructural localization studies of this protein. The antibody was obtained from an IgG fraction (AB 121) originally raised against the calcium binding protein calsequestrin by immunoabsorption on isolated rat liver hsp60. As shown by partial N-terminal amino acid sequence analysis of immunoprecipitated proteins AB 121 contained reactivities against hsp60, calsequestrin and the glycoprotein fetuin. In rat heart AB 121 recognized calsequestrin and hsp60. In human and rat liver the only reacting protein was hsp60. In rat erythrocytes the antibody bound to 61 kDa and 58 kDa isoforms of fetuin. According to published data no amino acid sequence homologies nor common motifs are found between calsequestrin, hsp60 and fetuin. As the first application the anti-hsp60 antibody was used for immuno-gold electron microscopical localization of hsp60: in myocardiocytes and hepatocytes of the rat strong labelling was obtained exclusively in mitochondria. No extramitochondrial structures were labelled. The specificity of the antibody and its ability to be visualized by immuno-gold electron microscopy offers the possibility to study the expression of this protein in the liver and in other organs. Possible clinical applications of these studies are discussed, since hsp60 could be a target antigen of autoantibodies in diseases such as autoimmune hepatitis, primary sclerosing cholangitis or primary biliary cirrhosis.

Amino Acid Sequence↗

PubMed1994

[Radiologic differential diagnosis of inflammatory round pulmonary infiltrates in immunocompromised patients. A prospective study using CT and MRT].

In a prospective study we examined the diagnostic ranking of CT and MR in 52 immunocompromised patients with nodular pulmonary lesions and clinical suspicion of invasive pulmonary aspergillosis (IPA). For early diagnosis of IPA (clinical symptoms having existed for less than 10 days) the CT halo sign proved highly sensitive and specific. MRT showed at this time a comparatively high sensitivity but only low specificity that could not be improved upon after Gd-DTPA. At a later stage of the aspergillosis infection (clinical symptoms manifested for more than 10 days) MR identified aspergillus-specific lesions with on-target characteristics (marked enhancement of margins after Gd-DTPA) or the so-called "reverse" target phenomenon (T2-weighted sequences). Such lesions were never seen in the early stage of the disease in patients with nodular pulmonary lesions of different aetiology (pseudomonal or staphylococcal pneumonia).

Adult↗

PubMed1994

[Therapy of invasive aspergillosis with itraconazole: our own experiences and review of the literature].

Invasive aspergillosis is one of the most threatening infections in immunocompromised patients of risk. The classical standard therapy of deep-seated mycoses--amphotericin B as monotherapy or in combination with 5-fluorocytosine--shows a response rate in aspergillosis of 55-60%. The new triazole compound itraconazole demonstrating a high in vitro activity against Aspergillus species was used in the recent years in several studies in the treatment of invasive aspergillosis. We report on our own experiences of itraconazole therapy in invasive aspergillosis with 20 patients suffering from well-known classical underlying diseases and factors of risk. Diagnosis was based on clinical symptomatology and was serologically and culturally proved. Special efforts were made for an early diagnosis according to clinical criteria. Therefore, in numerous cases clinical diagnosis was made several days prior to mycological confirmation, and antimycotic therapy could be started. The mean dosis administered was 400 mg itraconazole per day. With four patients no therapy response could be observed. In one patient a partial remission was obtained. In 15 patients aspergillosis was cured. In our patients the rate of complete remission, therefore, amounted to 75%. In evaluating 12 published studies on itraconazole therapy of invasive aspergillosis (including our own patients) a response rate of 63% (169 of 269 patients) could be calculated. Therefore, increasing experience is indicating that itraconazole therapy might be an alternative to classical amphotericin B therapy of invasive aspergillosis.

AIDS-Related Opportunistic Infections↗

PubMed1993

Oligosaccharide reprocessing and recycling of a cell surface glycoprotein in cultured rat hepatocytes.

The metabolism of the cell surface glycoprotein dipeptidyl peptidase IV(DPPIV) was studied in cultured rat hepatocytes. In pulse-chase labelling experiments using L-[35S]methionine a 100-kDa high-mannose precursor polypeptide is converted into the mature complex-type 110-kDa glycoprotein. Digestion with exo- and endoglycosidases and metabolic labelling with radioactive sugars demonstrate that the 110-kDa form contains about 6 complex-type oligosaccharides which are fucosylated and sialylated. About 25 min after the beginning of the pulse-labelled glycoprotein appears in the sinusoidal membrane. Physiologically only the 110-kDa form is found in the cell surface. If cell surface DPP IV was desialylated by sialidase at 4 degrees C, it is resialylated during incubation at 37 degrees C. This oligosaccharide reprocessing indicates that the surface glycoprotein has been recycled to the cell compartment containing terminal glycosyltransferases (presumably the trans Golgi system). Two different methods demonstrate internalization of cell surface DPP IV: 1) The complex cell surface DPPIV -anti-DPP IV-antibody -L-[35S]methionine-labelled secondary goat-anti-mouse-antibody formed at 4 degrees C becomes less accessible to trypsin during incubation at 37 degrees C. 2) Part of the complex plasma membrane DPP IV-anti-DPP IV-antibody formed in the cold cannot be recognized by the radioactive secondary antibody after rewarming. Internalization is not blocked by inhibition of protein synthesis with cycloheximide. During internalization of plasma membrane DPP IV its concentration in the membrane remains constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Inhibition of protein N-glycosylation by 2-deoxy-2-fluoro-D-galactose.

The effects of 2-deoxy-2-fluoro-D-galactose (dGalF) on N- and O-glycosylation of proteins was studied in rat hepatocyte primary cultures and in human monocytes. In hepatocytes, dGalF at concentrations of 1 mM or higher completely inhibited N-glycosylation of alpha 1-antitrypsin and alpha 1-acid glycoprotein, whereas 4 mM-2-deoxy-D-galactose (dGal) only slightly impaired N-glycosylation. In monocytes, 1 mM- or 4 mM-dGalF blocked N-glycosylation of alpha 1-antitrypsin and of interleukin-6, while O-glycosylation of interleukin-6 remained unaffected. In monocytes, dGal had no effect on protein N-glycosylation. Addition of uridine effectively prevented the UTP deficiency induced by dGalF, but had no effect on the inhibition of protein N-glycosylation by dGalF. Using 19F-n.m.r. spectroscopy, 2-deoxy-2-fluoro-D-galactose 1-phosphate (dGalF-1-P), UDP-dGalF and UDP-dGlcF could be identified as the major metabolites of dGalF in hepatocytes as well as in monocytes. In conclusion, compared with dGal, dGalF is a more efficient inhibitor of protein N-glycosylation. The effect is not caused by the depletion of UTP induced by dGalF, but rather by metabolites of dGalF. dGalF is metabolized not only in hepatocytes but also in peripheral blood monocytes, which can be used for ex vivo studies of disturbances in D-galactose metabolism.

Animals↗

PubMed1991

[Conservative therapy of ulcerative colitis and Crohn disease].

Basis treatment of severe ulcerative colitis and Crohn's disease comprises the systemic administration of corticosteroids. If the conditions are less severe, treatment with oral sulfasalazine or 5-aminosalicylic acid (5-ASA) can be attempted. Distal ulcerative colitis may respond to topical corticosteroids of enemas with sulfasalazine or 5-ASA. In long-term treatment with high-dose corticosteroids, azathioprine and 6-mercaptopurine may be employed to reduce the corticosteroid requirement. There ist no known generally applicable diet for the treatment or prophylaxis of these conditions.

Adrenal Cortex Hormones↗

PubMed1991

[Gastroenterologic findings in graft versus host disease after allogenic bone marrow transplantation].

We are reporting on a 25 years old patient with acute myelogenous leukemia, who developed an acute graft-versus-host disease (GVHD) 43 days after allogeneic bone marrow transplantation (BMT). The clinical symptoms included exanthema, diarrhea and abdominal cramps. The patient was treated with cyclosporine A and prednisone and the clinical symptoms disappeared subsequently. At day 225 post BMT the patient became icteric as the clinical manifestation of chronic GVHD. We describe in this case report endoscopical and histological findings during the episodes of acute and chronic graft-versus-host disease. The results obtained by sigmoidoscopy and liver biopsy confirmed the clinical diagnosis. The clinical work up of patients with acute or/and chronic GVHD should also include sigmoidoscopy in order to verify this transplantation related complication.

Adult↗

PubMed1990

Hepatocyte specific long lasting inhibition of protein N-glycosylation by D-galactosamine.

The effect of D-galactosamine on protein N-glycosylation was studied in rat hepatocyte primary cultures for alpha 1-antitrypsin (three complex type oligosaccharide chains) and alpha 1-acid glycoprotein (six complex type oligosaccharide chains). D-Galactosamine at a concentration of 4 mM inhibited partially de novo N-glycosylation leading to the formation of alpha 1-antitrypsin lacking one to two and of alpha 1-acid glycoprotein lacking one to five of its carbohydrate side chains. In addition D-galactosamine interfered with oligosaccharide processing, leading to the formation of some carbohydrate side chains remaining in an endoglucosaminidase H sensitive, i.e., not completely processed, form. D-Galactosamine impaired the secretion of alpha 1-antitrypsin and of alpha 1-acid glycoprotein but did not inhibit the secretion of the unglycosylated albumin. The inhibitory effect of D-galactosamine on de novo glycosylation as well as on oligosaccharide processing lasted for at least 24 h after it had been removed from the cells. D-Galactosamine impaired the glycosylation of alpha 1-antitrypsin only in hepatocytes, but not in human monocytes. Furthermore, D-galactosamine did not impair the N- and O-glycosylation of interleukin-6 in human monocytes and in MRC 5 fibroblasts. The results indicate that the effect of D-galactosamine on protein glycosylation is restricted to D-galactosamine metabolizing hepatocytes and is not exerted by the drug itself but by its metabolites.

Acetylglucosaminidase↗

PubMed1990

[Non-cirrhotic portal hypertension].

A chronic increase of the portal venous pressure is not only a sequel of liver cirrhosis. There is a large group of different diseases leading to "non-cirrhotic portal hypertension". Clinical presentation, diagnosis and treatment are discussed. The discrimination between cirrhotic and non-cirrhotic portal hypertension is important for the understanding of differences in clinical signs and course of the diseases, however.

Arteriovenous Fistula↗

PubMed1989

Effect of the alpha-glucosidase inhibitor N-hydroxyethyl-1-deoxynojirimycin (Bay m 1099) on the biosynthesis of liver secretory glycoproteins.

The effect of the alpha-glucosidase inhibitor N-hydroxyethyl-1-deoxynojirimycin (Bay m 1099) on the glycosylation and secretion of alpha 1-antitrypsin (three complex type oligosaccharide chains) and of alpha 1-acid glycoprotein (six complex type oligosaccharide chains) was studied in rat hepatocyte primary cultures. In the presence of 4 mM Bay m 1099 the processing of high-mannose to complex type oligosaccharides was partially inhibited leading to the secretion of alpha 1-antitrypsin and alpha 1-acid glycoprotein carrying a mixture of both high-mannose and complex type oligosaccharides. The major part of alpha 1-antitrypsin secreted by Bay m 1099 treated cells still carried two complex type oligosaccharide chains, the majority of alpha 1-acid glycoprotein carried three to five. Despite its effects on protein glycosylation Bay m 1099 did not lead to pronounced changes in the synthesis or secretion of alpha 1-antitrypsin, alpha 1-acid glycoprotein or albumin. At concentrations of Bay m 1099 lower than 0.5 mM no inhibitory effect on oligosaccharide trimming could be observed. After removal of Bay m 1099 from hepatocytes its inhibitory effect on protein glycosylation was immediately reversible.

1-Deoxynojirimycin↗

PubMed1989

[Dipeptidyl aminopeptidase IV in hospitalized patients and in galactosamine hepatitis of the rat: Activity and lectin affinity chromatography in serum and hepatic plasma membranes].

The activity of dipeptidyl aminopeptidase IV was studied in the sera of 378 hospitalized patients. The mean activity of dipeptidyl aminopeptidase IV was elevated significantly in patients with neoplasmata and hepatitis, but not in patients with liver cirrhosis. Significant correlations (p less than 0.001) existed with gamma-glutamyl transferase, glutamate dehydrogenase, alkaline phosphatase and leucine aminopeptidase. A significant correlation with lactate dehydrogenase existed only in patients with neoplasmata. Principal component analysis, performed with aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, leucine aminopeptidase, lactate dehydrogenase and dipeptidyl aminopeptidase IV, revealed correlations between the activities of aspartate aminotransferase and alanine aminotransferase, and between alkaline phosphatase and leucine aminopeptidase, but neither dipeptidyl aminopeptidase IV nor lactate dehydrogenase showed any correlation with either of these two groups. In lectin affinity chromatography with concanavalin A and wheat germ lectin sepharose, serum dipeptidyl aminopeptidase IV from liver cirrhosis patients showed the same binding pattern as that from healthy subjects. The activity and glycosylation of dipeptidyl aminopeptidase IV in serum and hepatic plasma membranes was investigated in rats, following the induction of hepatitis with galactosamine. In the serum, dipeptidyl aminopeptidase IV activity was elevated as early as 6 h after galactosamine injection, and the elevated activity persisted until the 7th day. At the same time dipeptidyl aminopeptidase IV activity was also elevated in the hepatic plasma membrane. Ninety eight percent of hepatic dipeptidyl aminopeptidase IV bound to concanavalin A as well as to wheat germ lectin and this value was unchanged during hepatitis. In the serum of control rats, 90% of dipeptidyl aminopeptidase IV bound to concanavalin A but only 39% to wheat germ lectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV.

The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.

Animals↗

PubMed1987

Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane.

Dipeptidyl peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns. The specific activity of the final preparation was 49.4 mumol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-polyacrylamide gel electrophoresis of the arginine-Sepharose eluate showed a single protein band with a molecular weight of 105,000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.

Animals↗