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Biomedical subjects

A Chrambach

Publications and source records attributed to A Chrambach.

At least 145 records · Page 8Linked to original sources
PubMed1981

Isolation of human growth hormone isohormones D and E in milligram amounts (I), using isotachophoresis on polyacrylamide gel.

Human growth hormone (hGH) isohormones D and E were isolated from a plasmin digest of 150 mg hGH. Isotachophoresis on a cylindrical polyacrylamide gel of 18 mm diameter served to separate the various isohormone species. The "extended stack" was sliced, and slices containing hGH-D and -E were extracted by Steady-State Stacking on 1% agarose gel and collected in a 1 ml volume. The extract was purified on gel chromatography and lyophilized. The overall yield was approximately 19 mg of hGH (D + E) and 16 mg of hGH-(D, minor E). This represents a 62% recovery of the amount of hGH-D and -E in the digest estimated densitometrically. The purity of the product, based on Lowry analysis, specific RIA or UV-absorbance, was 91%. The isolated hGH-(D+E) exhibited an enhanced bioactivity in the rat tibia line assay.

Animals↗
PubMed1981

Protein composition of plasmin preparation "homolysin".

A commercial preparation of human plasmin (Homolysin), capable of catalyzing the transformation of human growth hormone (hGH) into biologically activated species, was analyzed by electrophoresis and electrofocusing on polyacrylamide gel. Each major component of the preparation was characterized with regard to molecular size (retardation coefficient, KR), molecular net charge (y-intercept on the Ferguson plot, Y0), apparent isoelectric point (PI') and enzyme activity. The multiple components of Homolysin revealed by staining corresponded to various aggregation states of plasmin and exhibited full serine protease activity. Polyacrylamide gel electrophoresis of Homolysin in the presence of sodium dodecylsulfate (SDS) yielded 2 subunits which corresponded in molecular weight to the known plasmin subunits.

Dithiothreitol↗
PubMed1981

Isolation of human growth hormone isohormones D and E in milligram amounts (II), using isoelectric focusing on polyacrylamide gel.

Human growth hormone (hGH) isohormones D and E were prepared from a partial enzymatic hydrolyzate catalyzed by plasmin, using isoelectric focusing on polyacrylamide gel to fractionate the digest. Separation between the two isohormones species was optimized by employing a maximally flattened pH gradient between pH's 4.3 and 5.6. Gel slices containing isohormones D and E were extracted and concentrated by Steady-State Stacking on polyacrylamide gel. The extract was purified by gel filtration and lyophilized. Overall yields of lyophilized isohormones D and E were 10.9 mg and represented a recovery of 51% relative to the densitometrically estimated isohormone concentrations in the hGH digest. Purity of the products, based on Lowry analysis, U.V. absorbance and radioimmunoassay (RIA) was 60-70%. The isolated isohormone species were active in the rat tibia-line assay and in the specific RIA for hGH.

Acrylic Resins↗
PubMed1981

ONe-step isolation of human chopionic ganadotropin in milligram amounts, using selective steady-state stacking on polyacrylamide gel.

Human chorionic gonadotropin (hCG) has been isolated in a single step from a crude, commercially available urinary preparation. The isolated hormone, and highly purified standard hormone preparations, each exhibit on PAGE (pH 5.5), as on electrofocusing, 3 to 5 major "change isomeric" species within preparations. In addition, PAGE (pH 5.5) detects differences in molecular size between charge isomeric species of identical net charge occurring in the various preparations. For the purposes of isolation by Steady-State Stacking, each of these charge isomeric families of hCG components was collectively separated from faster migrating proteins in crude, commercial hCG by first allowing them to unstack while the more highly mobile contaminating proteins in the preparation remained stacked and were eluted as a stack from an elution-PAGE apparatus. After some time of electrophoresis the unstacked hCG components were then combined ("restacked") by sweeping them into a second moving boundary and eluted from a preparative elution-PAGE apparatus in a single zone. The product exhibits on PAGE re-run molecular species related to hCG standard as either "size-" or "charge-isomers". After dissociation into subunits, all preparations yield a protein indistinguishable in size or net charge from a standard preparation of the beta-chain of hCG. The alpha- and beta-chains derived from the isolated preparations exhibited dose-response curves parallel to those of standard hCG alpha and hCG beta respectively. The biological activity of the product was 8-12.000 IU/mg, as compared to 3,000 IU/mg for the starting material. The total yield was 20% by dry weight of the crude starting material.

Buffers↗
PubMed1981

Physical, biological, and immunological characterization of highly purified urinary human chorionic gonadotropin components separated by gel electrofocusing.

A highly purified urinary hCG preparation was subjected to electrofocusing on polyacrylamide gel. It was shown to fractionate into at least 11 components that can be detected by coomassie blue staining, a radioligand-receptor assay (RRA), and four hCG RIAs using antisera against the purified hCG, its alpha- and beta-subunits, and the hCG beta carboxyl-terminal peptide. Refocusing shows that these components are not artifacts of the electrofocusing technique. Six of the major components were isolated, and their pIs were shown to correlate with their sialic acid content. When these components were assayed by radioligand-radioreceptor assay, the four hCG RIAs, and a rat uterine weight assay, they showed 1) similar in vitro biological activities, 2) identical immunological activities that were indistinguishable from the in vitro biological activities, 3) hCG-like and not hLH-like immunological characteristics, and 4) different in vivo biological activities which increased with sialic acid content. Thus, sialic acid alone and/or differences in subunit assembly seem to be responsible for the electrophoretic heterogeneity of highly purified hCG.

Chorionic Gonadotropin↗
PubMed1980

A method for isolation by gel electrofocusing of isohormones B and C of human prolactin from amniotic fluid.

Human prolactin from amniotic fluid, consisting of isohormones B and C (major), was radio-iodinated after storage of the hormone for 3 years at -70 degrees C, and yielded a Ferguson plot in polyacrylamide gel electrophoresis that was indistinguishable from the original except that the zones of isohormone B and C were fused. However, isohormones B and C of 125I-labelled human prolactin were separated on isoelectric focusing in polyacrylamide gel, using Ampholine carrier ampholytes (pI range 5-8), taurine (pI 5.1) as anolyte and beta-alanine (pI 6.9) as catholyte. After 20 h of electrofocusing at 0-4 degrees C, 1000 V, both isohormones reached constant pH (isoelectric) positions on the gel. The apparent isoelectric points of human prolactin B and C were 5.96. Micro-preparative gel electrofocusing followed by excision and re-electrofocusing of the gel slices containing human prolactin B and C, yielded zones of homogeneous isohormones B and C.

Amniotic Fluid↗
PubMed1979

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents.

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.

Adenylyl Cyclases↗