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PubMedDate not supplied

The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory.

Acids↗
PubMed1975

The substructure of heavy meromyosin. The effect of Ca2+ and Mg2+ on the tryptic fragmentation of heavy meromyosin.

Heavy meromyosin, obtained by tryptic digestion of myosin, containing two main polypeptides whose masses were estimated as 81,000 and 74,000 dlatons from Na dodecyl-SO4 polyacrylamide gel electrophoresis, was further digested with trypsin. The Ca2+-activated ATPase activity remainded unchanged and the K+-EDTA activity increased while various smaller fragments were formed. The formation of some of these fragments is affected by Ca2+ or Mg2+ as first shown by Bálint et al. (Bálint, M., Schaefer, A., Biro, N. A., Menczel, L., AND Fejes, E. (1971) J. Physiol. Chem. Phys. 3, 455). On the basis of the time course of the appearance of fragments the following relationship emerges: see article. The 64K leads to 60K step is inhibited by divalent cations, while the breakdown of the 74K fragment is accelerated. The effect of Ca2+ was maximal at 0 similar to 0.1 muM, that of Mg2+ at 10 muM. The original light chains of myosin are not present in the heavy meromyosin serving as the starting material, but peptide material appears on electrophoresis in positions starting material, but peptide material appears on electrophoresis in positions where the light chains would be found. The fragments marked by an asterisk are considered to ba alpha-helical on the basis of their solubility at low ionic strength after precipitation with ethanol (Bálint et al.). The fact that alpha helical fragments are derived from the 60,000-dalton fragment indicateds that it is adjacent to the light meromyosin in the intact myosin while the 74,000- dalton fragment would be part of heavy meromysoin subfragment 1. Chromatography of Sephadex G-200 separates fractions with ATPase activity corresponding to heavy meromyosin and heavy meromyosin subfragment 1. Electrophoresis of these Sephadex fractions suggests that the main peptide constituting heavy meromysoin subfragment 1 is connected by noncobalent forces to a portion of the rod that is not immediately adjacent to it in the primary sequence. The significance of this finding is discussed in terms of the flexibility of the myosin head.

Adenosine Triphosphatases↗
PubMed2002

Analysis of DNA fragmentation in bovine somatic nuclear transfer embryos using TUNEL.

The production of cloned animals is an inefficient process because of early or late embryonic losses. This study focused on the DNA fragmentation that occurs during embryonic development. The occurrence of DNA fragmentation was examined in bovine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (NT) using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL). IVF and NT embryos at the two-cell to blastocyst stage were stained by TUNEL for the analysis of DNA-fragmented nuclei and with propidium iodide for determination of the total number of cells. DNA fragmentation was first detected in NT embryos at the four-cell stage, but in IVF embryos at the six- to eight-cell stage. The percentage of embryos with at least one DNA-fragmented nucleus increased with the advance of the developmental stage of embryos in both IVF and NT groups. The DNA-fragmented nucleus index in NT embryos that developed beyond the four-cell stage was significantly higher (P<0.01) than that of IVF embryos at the same stage. In the both IVF and NT groups, TUNEL-labelled cells were detected in almost all blastocysts and were mainly observed in presumptive inner cell mass (ICM) cells of embryos. The DNA-fragmented nucleus index was negatively correlated with the total number of cells in NT blastocysts, but this relationship was not observed in IVF blastocysts. These results suggest that the high occurrence of DNA fragmentation observed in NT embryos may be related to early embryonic loss after transfer.

Animals↗
PubMed2005

[Identification of human platelet specific functional antibody and its fragments].

OBJECTIVE: To study the immunoreactivity of the specific anti-platelet glycoprotein (GP) IgG antibody and its F(ab')2 fragments from patients with chronic idiopathic thrombocytopenic purpura (ITP) and to investigate their effects on platelet aggregation function. METHODS: Peripheral blood samples were collected from 84 patients with ITP. Modified monoclonal antibody immobilization of platelet antigen assays was used to detect the IgG antibodies specific for GP I b/II a, GP I b/IX and GP VI. The IgG antibody and its F(ab')2 fragments in the positive plasma inhibiting platelet aggregation function were prepared and purified. Plate-rich Peripheral blood sample was collected from a normal person with O type blood and platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared. Plasma pf ITP patient or purified IgG or F(ab')2 fragments of different concentrations were added into PRP, and then inducers of platelet aggregation ADP, ristocetin, or collagen were added. The platelet aggregation was measured. Platelet GP II b/III a specific human-rat chimeric antibody 7E3 and GP I b specific antibody SZ2 were used as positive controls and PBS was used as negative control. RESULTS: GP II b/III a and/or GP I b/IX and/or GP VI specific antibodies were found in 48 (57.1%) patients. The plasma, purified IgG and F(ab')2 fragments of 7 of these 48 patients (14.6%) with positive autoantibody showed significant activity against GP II b/III a (4 patients), GP I b/IX (2 patients), or GP VI (one patient). The purified IgG and F(ab')2 fragments of 2 patients positive in GP II b/ III a autoantibody out of the 7 patients significantly inhibited the platelet aggregation induced by ADP, the purified IgG and F(ab')2 fragments of 1 patients positive in GP I b/IX out of the 7 patients significantly inhibited the platelet aggregation induced by ristocetin, and the purified IgG and F(ab')2 fragments of 1 patients positive in GP VI out of the 7 patients significantly inhibited the platelet aggregation induced by collagen. CONCLUSION: A functional fragment, F(ab')2 portion of IgG is responsible for the autoantibody interaction with platelet GPs in ITP, and some of them also affect the platelet function. It can be used to develop completely humanized anti-GP small molecular phage antibody.

Adolescent↗
PubMed1991

Composition of proteoglycan fragments from hyaline cartilage produced by granulocytes in a model of frustrated phagocytosis.

An in vitro model of frustrated phagocytosis was developed in which granulocytes interact with well-defined slices of hyaline cartilage. The composition of the purified proteoglycan fragments released from the cartilage slices by N-formyl-methionyl-leucyl-phenylalanine-stimulated granulocytes was studied after 30, 60 and 90 min incubation time. It was shown that the proteoglycan fragments do not change their composition during incubation. The only change observed during incubation was an increase in the quantity of the fragments. The protein content of the proteoglycan fragments is 7.0-8.6%, corresponding to a peptide chain of 24-28 amino acids, and the relative molecular mass of the total fragment is Mr = 37,600-39,200. On average, each proteoglycan fragment contains two chondroitin sulphate chains (Mr = 22,000-22,400), every fourth fragment contains a keratan sulphate chain (Mr = 7000-7200) and every seventh to eighth contains an O-glycosidic oligosaccharide, whereas no N-glycosidic oligosaccharide could be detected. The results of the disaccharide analysis show that the galactosaminoglycan chains contain 76.2-83.6% chondroitin 4-sulphate, 12.9-19.4% chondroitin 6-sulphate, 3.5-3.8% chondroitin and no dermatan sulphate. Since composition and relative molecular mass of the chondroitin sulphate and keratan sulphate chains from the proteoglycan fragments resemble those of native proteoglycans, the conclusion may be drawn that the degeneration of the proteoglycans occurs by proteases that attack preferably the chondroitin sulphate-rich region of the core protein. This is the first inflammation model of joint destruction, which demonstrates the elution of soluble specific proteoglycan degradation products of defined size.

Amino Acids↗
PubMed1990

Biodistribution and radiation dose estimates for yttrium- and iodine-labeled monoclonal antibody IgG and fragments in nude mice bearing human colonic tumor xenografts.

An anti-carcinoembryonic antigen murine monoclonal antibody designated NP-4, and its F(ab')2 and Fab' fragments, were coupled to the 1/1 mixture of 1-isothiocyanato-benzyl-3-methyl- and 1-methyl-3-isothiocyanato-benzyl-diethylenetriaminepentaacetic acid chelate and labeled with 111In or 88Y. Biodistribution studies in nude mice bearing a human colonic tumor xenograft were performed with these labeled conjugates, and comparisons were made to unconjugated NP-4 IgG and fragments labeled with 131I. Regardless of the labeling method, higher tumor uptake was found with the intact IgG than with the fragments, but due to faster blood clearance, tumor/blood ratios were higher for the fragments than for the IgG. Tumor uptake for the radiometal-labeled NP-4 was generally higher than the 131I-labeled NP-4. Tumor/nontumor ratios for the liver, kidney, and spleen were higher for the 111In- and 88Y-labeled NP-4 IgG than the respective radiometal-labeled fragments, but tumor/nontumor ratios for the 131I-NP-4 fragments were higher than the 131I-NP-4 IgG. Radiometal uptake in the kidney was approximately 8 and 150 times higher than the 131I-NP-4 F(ab')2 and Fab', respectively, and the clearance of radiometal activity in the kidneys was approximately 10 times slower than the radioiodine. Quantitation of 88Y or 111In activity in the femur showed 3-5%/g for the IgG and F(ab')2 and only 1-2%/g for the Fab'. The amount of radioactivity in the femur remained constant over time, and between 60 and 100% of the 88Y activity remained after flushing the core of the femur with saline, whereas 50-70% of the 111In and only 25-30% of the 131I activity remained after washing. Radiation dose estimates derived from these studies suggest that at the maximal tolerated dose 131I-NP-4 IgG would deliver 5.9 times the dose to the tumor as 90Y-labeled NP-4 IgG. 90Y-labeled fragments would not be useful due to higher doses to the kidneys than to the tumor. However, with 131I-labeled IgG and fragments there is greater flexibility to permit tumoricidal doses without excessive toxicity to the normal tissues.

Animals↗
PubMed1988

Development of highly sensitive immunoassays to measure human chorionic gonadotropin, its beta-subunit, and beta core fragment in the urine: application to malignancies.

A variety of malignancies have been associated with the presence of human chorionic gonadotropin, hCG, its subunits, and fragments of its beta-subunit in blood and urine. The usefulness of these hCG-related tumor markers in nontrophoblastic malignancies has been inhibited by inadequate assay techniques. In order to achieve the required sensitivity and specificity, concentration steps and other procedures to remove cross-reacting human luteinizing hormone were necessary. In addition, the coexistence of a fragment of the hCG-beta or beta human luteinizing hormone subunit contributes to significant errors of measurement in urine. The importance of the hCG-beta fragment as a potential tumor marker has been recognized previously but no method was available to measure this antigen readily. We report here the development of a series of radioimmunometric, two-site assays which will accurately measure hCG, hCG-beta subunit, and the beta-subunit fragment directly in small volumes of unprocessed urine. These assays are highly specific, extremely sensitive, and not labor intensive since they employ microtiter plate procedures. Application of these assays to urine samples from patients with gynecological malignancies indicated that over 50% of all patients tested excreted the hCG-beta fragment in their urine. Also, this fragment comprised more than 50% of the moles of hCG immunoreactive components present in the specimens that were positive for hCG. This cancer marker is also demonstrable in trophoblastic malignant states such as choriocarcinoma in which the low molecular weight fragment can also be visualized directly by immunoblotting procedures. We conclude that a search for hCG immunoreactivity in the urine of patients with malignancies will be improved by the inclusion of accurate measurements of the prominent quantities of the beta fragment excreted by these individuals.

Biomarkers, Tumor↗
PubMed1989

Fragment A of diphtheria toxin causes pH-dependent lesions in model membranes.

Fragment A of diphtheria toxin has been shown to insert into lipid bilayers at low pH (Montecucco, C., Schiavo, G., and Tomasi, M. (1985) Biochem. J. 231, 123-128; Zhao, J.-M., and London, E. (1988) J. Biol. Chem. 263, 15369-15377). In this report, evidence is provided which demonstrates that fragment A, like diphtheria toxin, can also cause the release of a fluorescent dye (calcein) from vesicles under acidic conditions and that this release parallels fragment A insertion into the membrane. Although the permeability changes are not as large as those obtained with whole toxin (Jiang, G.-S., Solow, R., and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429), molecular sieving experiments indicate that the lesion induced by fragment A increases in size with decreasing pH and reaches an upper limit of 30 A at pH 4.0. In addition to size differences, the lesion induced by fragment A releases calcein in a graded manner, whereas diphtheria toxin causes an all-or-none release. One possible interpretation of this result is that the fragment A lesion is transient in comparison to that induced by whole toxin. Although the molecular bases for the observed differences are not understood, these data suggest that fragment A interaction with the lipid bilayer may play a significant role in mediating its own translocation across membranes and that fragment B may aid this process by initiating, enlarging, and stabilizing the lesion formed.

Calcium↗
PubMed1988

Characterization of an apparently lower molecular weight gamma-chain variant in fibrinogen Kyoto I. The replacement of gamma-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site.

Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal gamma-chain and a gamma-chain variant (gamma Kyoto I) that has an apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal Mr in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified gamma-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that gamma Asn308 of the gamma-chain variant is replaced by lysine. Purified fragment D1 of fibrinogen Kyoto I also contains two types of D1 gamma-remnants: normal and apparently lower Mr types. Abnormal fragment D1 is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) than normal fragment D1, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-gamma-chain monoclonal antibody. Analysis of peptides released from fragment D1 by plasmin in the presence of EGTA demonstrated the cleavage of the gamma Lys308-Gly309 bond. Fragment D1 of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing gamma Asn308 affects the polymerization of fibrin monomers and that the gamma Asn308----Lys replacement causes a conformational change in the gamma-chain which results in the accelerated cleavage of gamma Lys356-Ala357 and gamma Lys302-Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and Gly309 in the presence of EGTA. During these studies, we found that part of the gamma Lys212-Glu213 bond in fragment D1 is cleaved by plasmin in the presence of EGTA.

Amino Acid Sequence↗
PubMed1985

Proteolytic fragments identified with domains of the aspartate chemoreceptor.

Two proteolytic fragments generated during the preparation of the aspartate receptor from Salmonella typhimurium have been purified. These fragments are the products of a single cleavage by an endogenous protease after amino acid 259 in the sequence of the intact receptor. Proteolytic fragment 1 (PF1) represents amino acids 1-259 (Mr = 29,000); this unit retains the aspartate-binding function of the intact receptor. The second fragment (PF2) includes residues 260-552 (Mr = 31,000) and has the normal sites of reversible methylation for the receptor. Like the purified intact receptor, this fragment can be methylated in vitro, although at a much slower rate. Circular dichroic measurements suggest that both proteolytic fragments contain substantial alpha-helical structure, approximately 95 and 53% for PF1 and PF2, respectively. No beta-structure could be detected in either fragment. Molecular sieve chromatography in the presence of detergent suggests that PF1 occurs as a stable multimer of an order equivalent to that observed for the detergent-solubilized aspartate receptor, i.e. a tetramer (+/- 1). PF2 is found to have a multimeric form which is sensitive to the removal of detergent. It is proposed that these fragments represent structural and functional domains of the aspartate receptor.

Amino Acid Sequence↗
PubMed1986

Identification of functional domains on von Willebrand factor by binding of tryptic fragments to collagen and to platelets in the presence of ristocetin.

With the use of monoclonal antibodies that inhibit the ristocetin-induced binding of von Willebrand factor (VWF) to platelets and the binding to collagen, we have previously identified two distinct tryptic fragments. To prove that these fragments contain the platelet binding or the collagen binding domain, we investigated the direct binding of tryptic fragments of 125I-VWF to platelets in the presence of ristocetin and to collagen fibrils. During the course of the tryptic digestion, there was a rapid and parallel decrease in binding to platelets and collagen. In the first ten minutes, binding decreased greater than 50%; a further decrease to 19% and 29%, respectively, was noted at 90 minutes, but no further decrease was observed thereafter. The bound fragments were eluted from platelets and collagen and analyzed on polyacrylamide gradient gels. The fragments bound to the platelets appeared to be reduced, probably by endogenous reducing substances from the platelets. This was prevented by addition of N-ethylmaleimide during the incubation. After 24 hours of digestion, platelets predominantly bound fragments of 116 kd and collagen bound a single fragment of 48 kd. These fragments are similar to those previously identified with the monoclonal antibodies.

Binding Sites↗
PubMed1986

A staphylococcal alpha-toxin fragment. Its characterization and use for mapping biologically-active regions of alpha-toxin.

A fragment (alpha-13) of Staphylococcal alpha-toxin was compared with intact alpha-toxin as regards biochemical and biological properties, and the resulting information was used for mapping biologically-active regions of alpha-toxin. The alpha-13 fragment had an apparent Mr of 18,500, judged by sodium dodecylsulphate polyacrylamide gel electrophoresis. However, it showed the same relative mobility as alpha-toxin when subjected to gel filtration on Biogel P60, high pressure liquid chromatography or electrophoresis on polyacrylamide gradient gel. The fragment had roughly the same specific hemolytic activity as intact alpha-toxin. In contrast to intact alpha-toxin, the fragment was neither membrane-damaging to mouse adrenocortical (Y 1) tumour cells nor lethal to mice. However, a short treatment of Y 1 cells with the fragment completely blocked intoxication by subsequently-added alpha-toxin. Likewise, the lethal effect of alpha-toxin was inhibited when the fragment was injected prior to the toxin. Thus, the fragment had lost the active region(s) responsible for Y 1 cell intoxication and lethality, while the region(s) for binding to these targets, as well as the region responsible for hemolysis, were retained. On the basis of these findings and previous reports concerning tryptic fragments of alpha-toxin, a hypothetical map of the different biologically-active regions of alpha-toxin was established.

Animals↗
PubMed1987

Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly.

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.

Adrenal Cortex↗
PubMed1985

Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain.

CRM197, CRM176, and CRM228 are products of single or multiple missense mutations in the diphtheria toxin gene. CRM197 differs from wild-type toxin in 1 amino acid residue of the fragment A region, and also CRM176 and CRM228 have amino acid substitution(s) in fragment A. We compared the binding properties of CRM197 to toxin-sensitive Vero cells with those of diphtheria toxin and other CRMs. Nicked CRM197 is about 50 times more effective than intact CRM197 in inhibiting the action of diphtheria toxin on sensitive cells, as shown by inhibition of diphtheria toxin cytotoxicity or inhibition of binding of 125I-diphtheria toxin. The binding of native toxin or other CRMs was not significantly affected by nicking. Moreover, the binding of CRM197 to cells was unaffected by ATP, although ATP clearly inhibits binding of diphtheria toxin, CRM176, and CRM228. Two kinds of hybrid protein were formed using fragment B of CRM197: one with fragment A of diphtheria toxin and one with fragment A of CRM228. ATP inhibited the binding of these hybrid proteins. Furthermore, the affinities of these hybrid proteins for diphtheria toxin-sensitive cells were the same as that of native toxin. Thus, it was concluded that the altered binding properties of CRM197 were due to alteration of fragment A and what the interaction of diphtheria toxin with ATP involves both fragments. The results also suggest that fragment A plays a role in diphtheria toxin-receptor interaction.

Adenosine Triphosphate↗
PubMed1981

[Comparative study of substance P and its fragments: analgesic properties, effect on behavior and monoaminergic processes].

The effect of substance P (SP) and of its fragments 5-11, 8-11, 9-11, 10-11 administered into the brain ventricles in doses of 5, 25 and 50 nM on the behavior and content of biogenic monoamines of the rat brain was studied. The analgetic properties of the substances under consideration and those of fragment SP 10-11 in doses of 5, 25, 50 and 100 nM were also subjected to examination. It was found that SP and fragment 5-11 stimulate and enhance the locomotor activity in rats, while fragments 8-11 and 9-11 provoke hypoactivity. The substances under study increase the serotonin and dopamine turnover, whereas SP and fragment 8-11 lower the serotonin content as well. After administration of SP and fragment 5-11 analgesia was seen to transform to hyperalgesia depending on the dose. Fragments 8-11 and 9-11 produce analgetic effect. It is suggested that both SP fragments and the whole SP molecule can influence the neurochemical process that regulate behavior and pain perception.

Analgesics↗
PubMed1983

Insulin metabolism in rat hepatocytes. Evidence for generation of an insulin fragment missing a portion of the B chain involved in receptor binding.

Insulin metabolism by isolated rat hepatocytes was studied, utilizing A14 [125I]monoiodoinsulin, [3H]PheB1 semisynthetic insulin, and [3H]insulins synthesized by rat islets. Degradation was assessed by gel filtration, polyacrylamide gel electrophoresis, precipitation by anti-insulin antibody, and binding to specific insulin receptors on IM-9 human lymphocytes. When incubations were performed at 15 degrees C or less, insulin bound to hepatocytes remained intact for up to 2 h. At 37 degrees C we detected the generation of an insulin fragment with an apparent molecular weight of approximately 5000 whose electrophoretic mobility was greater than that of insulin. The fragment bound well to anti-insulin antibodies but poorly to insulin receptors. Information about the structure of the fragment was obtained by comparing the metabolism of [3H]PheB1 semisynthetic insulin with that of [3H]PheB1,24,25 insulin. The data suggest that the fragment contains PheB1 but is missing the PheB24 and PheB25. Treatment of the fragment with trypsin and carboxypeptidase B did not affect its electrophoretic mobility indicating that the fragment is also missing ArgB22. Incubation in the presence of 0.1 mM chloroquine led to accumulation of both intact insulin and the insulin fragment, suggesting that both are degraded by lysosomes. The results of this study suggest the presence of two pathways for insulin degradation in liver: a chloroquine-insensitive pathway by which a portion of the B chain consisting of at least 10 amino acids is removed and a chloroquine-sensitive pathway by which both insulin and the fragment are degraded.

Animals↗
PubMed1983

Hormonal control of glycogen synthase in rat hemidiaphragms. Effects of insulin and epinephrine on the distribution of phosphate between two cyanogen bromide fragments.

The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with [32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (Mapp = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the [32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of [32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 microM) inactivated glycogen synthase and increased its content of [32P]phosphate by about 50%. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The larger fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased [32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of [32P] phosphate present in Fragments I and II by about 40%. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the enzyme.

Animals↗
PubMed1982

On a domain structure of colicin E1. A COOH-terminal peptide fragment active in membrane depolarization.

A prolonged digestion of colicin E1 with dilute trypsin results in the generation of a trypsin-resistant peptide fragment of the colicin which is approximately one-third of the molecule. The amino acid composition of the fragment, Mr = 20,000, is more nonpolar in nature than the colicin, with the major change in composition being the reduction of the arginine content from 25 residues in the Mr = 56,000 colicin molecule to approximately 1 residue in the fragment. The NH2-terminal amino acid sequence of the tryptic fragment shows no similarity to that of colicin E1. Alignment of this sequence with the complete amino acid sequence of the colicin E1 molecule obtained by others, shows that the fragment occupies all, or almost all, of the COOH-terminal section of the molecule. The fragment behaved similarly to colicin E1, in being able to dissipate a potassium diffusion potential in unilamellar membrane vesicles made of dimyristoylphosphatidylcholine. The fragment was able to dissipate the diffusion potential above and below the temperature region (Tm = 23.5-24 degrees C) of the lipid phase transition in these vesicles, consistent with a channel-like function. The fragment did not show measurable binding to colicin receptor sites on the cell surface, but was much more efficient than colicin E1 in its ability to inhibit proline transport by inner membrane vesicles of Escherichia coli. These data imply that a membrane channel-forming function of the molecule is located in this 20,000 molecular weight region at the COOH-terminal end of the colicin molecule.

Amino Acid Sequence↗