scholarly journals Oxidation of polymines by diamine oxidase from human seminal plasma

1975 ◽  
Vol 145 (2) ◽  
pp. 373-378 ◽  
Author(s):  
E Hölttä ◽  
P Pulkkinen ◽  
K Elfving ◽  
J Jänne
Keyword(s):  
Molecular Weight ◽  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Diamine Oxidase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Semen ◽  
Human Seminal Plasma ◽  
Ph Values

1. Diamine oxidase [amine-oxygen oxidoreductase (deaminating)(pyridoxal-containing), EC 1.4.3.6] was purified from human seminal plasma more than 1,700-fold. The enzyme appeared to be homogeneous on polyacrylamide-gel electrophoresis at two different pH values. 2. The general properties of the enzyme were comparable with those described for other diamine oxidases from different mammalian sources. The molecular weight of the enzyme was calculated to be about 182,000. 3. The enzyme had highest affinity for diamines, but polyamines spermidine and spermine were also degraded at concentrations that can be considered physiological in human semen. 3. The possible degradation of spermine by diamine oxidase in human semen in vivo may give rise to the formation of cytotoxic aldehydes that conceivably can influence the motility and survival of the spermatozoa.

scholarly journals Vitellogenesis in the stick insect Carausius morosus I. Specific protein synthesis during ovarian development

1980 ◽  
Vol 46 (1) ◽  
pp. 1-16
Author(s):  
F. Giorgi ◽  
F. Macchi
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Fat Body ◽  
Polyacrylamide Gel Electrophoresis ◽  
Stick Insect ◽  
Polyacrylamide Gel ◽  
Carausius Morosus

Vitellogenesis in the stick insect Carausius morosus (Br.) has been studied with the goal of identifying vitellogenin in various tissues. Following exposure to in vivo to radioactive amino acids, oocytes in the medium size range are labelled with a minimum delay of 6 h after the time of injection. Incorporation of radioactivity under these conditions is shown to depend upon accumulation of proteins rather than on a differential rate of protein synthesis in succeeding stages of oogenesis. By immunochemical analyses, it is shown that at least two antigens are common to both haemolymph and ovary and that one of these is also present in the fat body. Both antigens are labelled during exposure to radioactive amino acids. When analysed by the SDS polyacrylamide gel electrophoresis, extracts from both haemolymph and ovary appear to share a number of protein fractions which range in molecular weight from 40 000 to 200 000 Daltons. The labelling pattern exhibited by these fractions is clearly indicative of a protein transfer from the fat body to the oocyte. Fat body cultured in vivo for up to 4 h releases a major macromolecular complex in the external medium. The latter has been identified as vitellogenin by both immuno-precipitation assay and SDS polyacrylamide gel electrophoresis. The protein which is synthesized and secreted under these conditions results from the processing of a protein complex of higher molecular weight.

scholarly journals Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity

1976 ◽  
Vol 155 (3) ◽  
pp. 679-687 ◽  
Author(s):  
M J Crabbe ◽  
R D Waight ◽  
W G Bardsley ◽  
R W Barker ◽  
I D Kelly ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Substrate Specificity ◽  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Diamine Oxidase ◽  
Amine Oxidase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Lysine Vasopressin

1. Isoelectric focusing studies of human placental diamine oxidase showed the pI value of the active enzyme to be 6.5. This information was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH gradient elution and this, together with affinity chromatography on concanavalin A-Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration of diamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultracentrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx. 1.0 and 1.2g-atom respectively/70000mol.wt. unit. 5. The e.s.r. spectral intensity did not decrease nor did the spectral line shape change when excess of p-dimethylaminomethylbenzylamine was added to the enzyme. 6. Addition of K13CN to the enzyme eliminated the copper e.s.r. signal without affecting the manganese signal. 7. The placental enzyme therefore appears to differ from other amine oxidases in terms of its metal cofactor requirement, molecular weight and substrate specificity, and possible roles in vivo for this enzyme are discussed.

In Yivo Behavior of Human Fibrinogen and Fragments X, D and E.

1975 ◽  
Author(s):  
Nicole Ardaillou ◽  
Lise Dray ◽  
Marie-José Larrieu
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Fibrinogen ◽  
Fraction I ◽  
Stage 1 ◽  
Lower Slope

Fragments X, D and E were obtained from plasmin digestion of purified human fibrinogen (fraction I-O). Fragment × was isolated from stage 1 digest by filtration on Sephadex G-200 by Budzinski and Marder, and fragments D and E in our laboratory by Pevikon block electrophoresis. The purified fragments were identified by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. Human fibrinogen and fragments X, D and E were labelled with 125iodine by the lactoperoxidase method. Plasma disappearance curves were studied in rabbits. A two exponential curve was obtained with all of them, with second exponential (lower slope) being clearly predominant. The half-lifes of the second exponentials were different for each fragment: there appears to be a relationship between the molecular weight of the fragments and their half-lifes. Values observed (mean±s.e.m.) were: fibrinogen 49.34±5.4 hours, fragment X 6.14±0.44 hours, fragment D 2.25±0.20 hours and fragment E 1.53±0.13 hours. No fragment was found either to polymerize or to bind to plasma proteins in vivo following injection into rabbits as demonstrated by the elution peaks obtained after filtration through Sephadex G-200.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

1984 ◽  
Vol 101 (1) ◽  
pp. 27-32 ◽  
Author(s):  
F. Mena ◽  
G. Martínez-Escalera ◽  
C. Clapp ◽  
C. E. Grosvenor
Keyword(s):  
Pituitary Gland ◽  
Incubation Period ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pituitary Glands ◽  
H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

scholarly journals Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin

1980 ◽  
Vol 191 (3) ◽  
pp. 799-809 ◽  
Author(s):  
R G Sutcliffe ◽  
B M Kukulska-Langlands ◽  
J R Coggins ◽  
J B Hunter ◽  
C H Gore
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Carbohydrate Content ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Tryptic Fragment

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

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