20 alpha-Hydroxysteroid dehydrogenase and 17 beta-estradiol dehydrogenase localize in cytosol of human term placenta

1982 ◽  
Vol 242 (3) ◽  
pp. E178-E183
Author(s):  
R. C. Strickler ◽  
B. Tobias
Keyword(s):  
Electron Microscopy ◽  
Dehydrogenase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Subcellular Fractions ◽  
Enzyme Assays ◽  
Marker Enzyme ◽  
Differential Centrifugation ◽  
Experimental Conditions ◽  
Human Term Placenta ◽  
Term Placenta

The 20 alpha-hydroxysteroid dehydrogenase activity in human term placenta has been localized by different investigators to nuclear, mitochondrial, microsomal, and cytosolic subcellular fractions. Furthermore, in the cytosol, 20 alpha-hydroxysteroid dehydrogenase activity may be a second function of the enzyme that mediates 17 beta-estradiol dehydrogenase activity. To search for a unique 20 alpha-hydroxysteroid dehydrogenase, human placental villous tissue, homogenized in three different buffer systems, was fractionated by differential centrifugation, and the 17 beta- and 20 alpha-activities were measured by radioisotope conversion assay. The enrichment and purity of the subcellular fractions were shown by marker enzyme assays and electron microscopy studies. Under all experimental conditions, 20 alpha-hydroxysteroid dehydrogenase activity was identified only in the 105,000 g placental cytosol: intact, osmotically ruptured, and acetone-extracted mitochondria, nuclei, and microsomes did not convert progesterone to 20 alpha-dihydroprogesterone. Furthermore, because 17 beta-estradiol dehydrogenase activity was in large part soluble in the cytosol, these localization studies are consistent with the hypothesis that the 20 alpha- and 17 beta-oxidoreductase activities in human placenta reside on one soluble protein.

THE SUBCELLULAR DISTRIBUTION OF 17β-HYDROXYSTEROID DEHYDROGENASE IN THE HUMAN TERM PLACENTA

1976 ◽  
Vol 82 (1) ◽  
pp. 150-163 ◽  
Author(s):  
C. M. G. Thomas ◽  
J. H. Veerkamp
Keyword(s):  
Dehydrogenase Activity ◽  
Subcellular Distribution ◽  
Medium Composition ◽  
Subcellular Fractions ◽  
Differential Centrifugation ◽  
Marker Enzymes ◽  
Cytosol Fraction ◽  
Human Term Placenta ◽  
Term Placenta ◽  
Buffer Medium

ABSTRACT Human term placenta tissue homogenates were subjected to differential centrifugation procedures. The composition of the subcellular fractions was monitored with a number of marker enzymes and the effectiveness of these enzyme systems was evaluated. The subcellular fractions were tested for their 17β-hydroxy dehydrogenase activity on testosterone, oestradiol and the synthetic substrate retrotestosterone. Buffer medium composition showed a direct influence upon enzyme distribution patterns of all fractions during the same differential centrifugation procedure. All enzyme activities tested became less sedimentable when glycerol was present in the fractionation buffer. Glycerol stabilized soluble 17β-hydroxy dehydrogenase activity during fractionation. The activity of steroid-converting enzymes was inhibited by the presence of glycerol in the medium. Subcellular distribution of marker enzymes did not sustain the presence of mitochondrial 17β-hydroxy dehydrogenase but related it to microsomal contamination. In general the proportion of 17β-hydroxy dehydrogenase activity in the particulate fractions showed a decrease in the substrate order retrotestosterone > testosterone > oestradiol which was independent from the buffer medium used. Specific activities for both particle-bound and soluble 17β-hydroxy dehydrogenase increased in the substrate order retrotestosterone < testosterone < oestradiol. The particulate enzyme activity was maximal with NAD+ for the three substrates tested, but in the cytosol fraction NADP+ was the preferential co-enzyme only when oestradiol was used as the substrate.

scholarly journals A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain

1978 ◽  
Vol 176 (2) ◽  
pp. 365-370 ◽  
Author(s):  
R F G Booth ◽  
J B Clark
Keyword(s):  
Electron Microscopy ◽  
Rat Brain ◽  
Rapid Method ◽  
Sucrose Gradient ◽  
Enzyme Assays ◽  
Marker Enzyme ◽  
Flotation Technique

A rapid (less than 2h) method is described for the preparation of synaptosomes from rat brain by using a discontinuous Ficoll/sucrose gradient by a flotation technique. These synaptosomes are metabolically active and minimally (less than 5%) contaminated with ‘free’ mitochondria as judged by marker-enzyme assays and electron microscopy.

scholarly journals Sialic acid in crude myelin fractions from rat brain

1975 ◽  
Vol 148 (3) ◽  
pp. 375-380 ◽  
Author(s):  
O K Langley
Keyword(s):  
Electron Microscopy ◽  
Sialic Acid ◽  
Rat Brain ◽  
Satellite Cell ◽  
Plasma Membranes ◽  
Enzyme Assays ◽  
Marker Enzyme ◽  
Cellular Membranes ◽  
Cell Plasma ◽  
Ultrastructural Appearance

Protein- and lipid-bound sialic acid was assayed in myelin fractions isolated by four different methods from rat brain homogenates. The extent to which non-myelin cellular membranes contaminate these fractions was assessed by electron microscopy and marker-enzyme assays. Small amounts of sialic acid found in the least contaminated myelin fractions are considered to be constituents of axonal and satellite cell plasma membranes known to be present. The data are discussed with reference to the ultrastructural appearance of myelin.

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