T.T. Tchen

Phospholipids of adrenal cortex mitochondria and the steroid hydroxylases: The lipid-environment of cytochrome P-450

Summary Adrenal cortex mitochondria have a high content of phospholipids with elevated ratios of phosphatidyl ethanolamine and phosphatidyl choline to diphosphatidyl glycerol, as compared with those of heart, liver, and kidney mitochondria. An extracted cytochrome P-450 preparation contained phosphatidyl ethanolamine and phosphatidyl choline as major components but no diphosphatidyl glycerol. After phospholipase-C pretreatment of the P-450 preparation, the reconstituted steroid 11 β-hydroxylase activity was inhibited, whereas the cholesterol desmolase activity was activated to a comparable extent.

Tyrosinase of the goldfish Carassius auratus L. I. Radio-assay and properties of the enzyme.

Abstract 1. 1. A sensitive radio-assay for tyrosinase was developed. 2. 2. The partial purification of the soluble tyrosinase in the skin of xanthic goldfish is reported. 3. 3. Some of the properties of the enzyme are described. 4. 4. Free dopa has been excluded as an obligatory intermediate in the oxidation of tyrosine by tyrosinase.

Some effects of Ca2+, Mg2+ , and Mn2+ on the ultrastructure, light-scattering properties, and malic enzyme activity of adrenal cortex mitochondria.

Abstract The ultrastructure and 90 ° light-scattering capacity of adrenal cortex mitochondria have been examined under conditions which lead to an activation of malic enzyme activity in these mitochondria. After isolation, the mitochondria display an aggregate ultrastructure which does not resemble the vesicular (orthodox) form normally seen in vivo . Under conditions of malic enzyme activation (presence of malate, NADP + , Mg 2+ and 1 m m Ca 2+ ), the ultrastructure reverts to a vesicular form as seen in vivo . Of these required components, only Ca 2+ affects the ultrastructure. The ultrastructural transformation from the aggregate to the orthodox form is always accompanied by a decrease in the 90 ° light-scattering capacity. When produced by Ca 2+ , transformation requires energy-dependent Ca 2+ uptake if an oxidizable substrate is present. In the absence of substrate, the transformation occurs as an apparent energy-independent effect. Mn 2+ can substitute for Ca 2+ only in the presence of substrate. In de-energized mitochondria, Mn 2+ prevents the effects of Ca 2+ . The activation of malic enzyme is always preceded by a decrease in light scattering and transformation to the orthodox ultrastructure; however, the presence of the orthodox form is not a sufficient condition since subsequent chelation of free Ca 2+ fails to reverse either the decrease in light scattering or ultrastructural transformation but does reverse the enzyme activation. In addition, levels of Mn 2+ which effectively depress light-scattering capacity and produce the orthodox form, fail to activate malic enzyme significantly. The data are discussed as they relate to Ca 2+ -induced damage in mitochondria.

Changes in some biochemical parameters including cytochrome P-450 after hypophysectomy and their restoration by acth administration in rats four months post hypophysectomy☆

Abstract Following withdrawal of ACTH by hypophysectomy, rat adrenal gland P450 content falls to a low level with a half life time of 3.5 days. The content can be restored by subsequent ACTH treatment in animals as long as four months post hypophysectomy with parallel regeneration of in vitro steroid synthetic capacity. The recovery pattern of a number of other gland parameters suggests that the regeneration of P450 and steroidogenic capacity in these severely atrophied glands occurs by a mechanism which is largely dependent on cell replication.

The role of Ca2+ in control of malic enzyme activity in bovine adrenal cortex mitochondria

Abstract Low physiological levels of Ca 2+ , in the presence of Mg 2+ allow the reduction of extramitochondrial NADP+ via intramitochondrial malic enzyme. The rate of reduction is dependent on the concentration of Ca 2+ and Mg 2+ . The Ca 2+ levels producing the appearance of malic enzyme activity also cause an ultrastructural transformation from the aggregated to the orthodox form. The phenomenon requires electron transport and is blocked by agents which interfere with active Ca 2+ accumulation.

Putrescine — α-Ketoglutarate transaminase in E. coli

Abstract A mutant of E . coli B was obtained which can grow on putrescine as the sole C and N source. This mutant converts putrescine to succinate via γ-aminobutyraldehyde, γ-aminobutyrate and succinic semialdehyde in similar manner as found in Pseudomonas fluorescens . The first reaction, however, was not catalyzed by a diamine oxidase, but by a transaminase which is constitutive in this strain of E . coli .

Isolation of xanthophores from the goldfish (Carassius auratus L.)

SummaryA method is described for the isolation of milligram quantities of viable, hormone-responsive xanthophores from goldfish scales. The preparations are typically 70 to 90% pure and are useful for biochemical analyses or for establishing primary cultures.

Hormone-induced filopodium formation and movement of pigment, carotenoid droplets, into newly formed filopodia

SummaryTreatment of cultured goldfish xanthophores by hormone (ACTH) or c-AMP induces not only pigment dispersion, but subsequent outgrowth of processes, and pigment translocation into these processes. These latter effects are shown to proceed as follows: First the edge of the cytoplasmic lamellae takes on a scalloped contour with numerous protrusions. These presumably serve as nucleation centers where short microfilament bundles are assembled, Later, the microfilament bundles elongate (“grow”), often resulting in an extension of the protrusions to become filopodia while the proximal end of the microfilaments penetrates into the thicker portion of the cellular process which now houses the pigment, i.e., the carotenoid droplets. Carotenoid droplets appear to migrate along the microfilament bundles, or cytoplasmic channels associated with them, into the filopodia. Finally, some of the filopodia become broader, thicker and laden with carotenoid droplets and are then recognized by light microscopy as pigmented cellular processes. The microfilaments have been shown to be actin filaments by their thickness, the size of their subunits, and decoration by heavy meromyosin. Evidence is presented which suggests that the growth of these actin filaments may come about by recruitment from short F-actin strands found in random orientation in adjacent areas.

Tyrosinase of the goldfish Carassius auratus L: II. Correlation of tyrosinase activity with pigmentation

1. n1. It has been demonstrated that during stress-induced melanogenesis in the goldfish, Carassius auratus L, the level of tyrosinase in the skin is increased several fold and that this increase precedes pigmentation. n n2. n2. The increase in tyrosinase activity is more pronounced in the particulate fraction than in the soluble fraction. n n3. n3. In three varieties of the goldfish (xanthic, wild-type grey and black moor), the tyrosinase activity parallels the degree of pigmentation of the skin. This is due to a higher level of particulate tyrosinase in the pigmented fish. n n4. n4. Considerable tyrosinase activity is present in skin homogenates prepared from totally xanthic fish. Therefore, in addition to the level of tyrosinase activity, other factors may regulate melanogenesis.

Intermediate filaments with novel protein composition from certain goldfish cells

Using the conditions for vimentin filament recycling, intermediate filaments (approximately 10 nm) were prepared from the cytoskeleton of a goldfish tumor cell line (erythrophoroma or xanthophoroma). 2-D analysis showed unusual protein composition, with four proteins of molecular weights of 60, 45, 56 and 51 kilodaltons in ratios of approximately 4:4:1:1. These correspond to four of the major cytoskeletal proteins of both the tumor cells and normal xanthophores.