Swainsonine, a potent mannosidase inhibitor, elevates rat liver and brain lysosomal alpha-D-mannosidase, decreases Golgi alpha-D-mannosidase II, and increases the plasma levels of several acid hydrolases

Swainsonine, a toxic plant alkaloid reported to be the agent that induces in animals a neurological condition very similar to the hereditary lysosomal storage disease mannosidosis, and to inhibit the formation of complex glycoproteins of the asparagine-linked class, was recently shown [D.R.P. Tulsiani, T.M. Harris, and O. Touster, (1982) J. Biol. Chem. 257, 7936-7939] to be a highly potent and specific inhibitor of Golgi mannosidase II in addition to being a strong inhibitor of lysosomal mannosidase. In the present study the effect of administered swainsonine on tissue enzyme levels was investigated. The activity of Golgi mannosidase II was markedly decreased (22% of control) without changes occurring in the activities of several other Golgi enzymes. However, the effects of swainsonine on lysosomal enzymes was unexpected. In liver, acid mannosidase increased markedly, instead of decreasing as would be expected from a compound reported to induce a mannosidosis-like condition. Similarly, the principal change in brain was a substantial increase in lysosomal mannosidase levels. In plasma, most lysosomal enzymes increased. These results indicate that the pathological effects of swainsonine are not solely attributable to its being an inhibitor of lysosomal alpha-D-mannosidase and are probably a consequence of abnormal processing of glycoproteins.     

Novel features of hamster pinealocyte ultrastructure

Pineal glands from young adult hamsters (Mesocricetus auratus Water-house) kept on a 12 : 12 light : dark photoperiod were examined by electron microscopy. Ultrastructural features of nuclei, mitochondria, and centrioles that have not been reported previously are presented in this paper. Of particular interest is the presence of modified centrioles in normal hamster pinealocytes.     

Partial purification and characterization of insulin protease and its intracellular inhibitor from rat liver

Insulin protease was purified 700-fold from rat liver homogenate by combined ultracentrifugation, ammonium sulfate fractionation, and glucagon-Sepharose-4B affinity chromatography. Optimum degradation of insulin was observed at pH 7.6 with the purified protease whose Km was 24 nM. The enzyme activity was inhibited completely by N-ethylmaleimide, p-hydroxymercuribenzoate, and heavy metals at 1 mM, whereas at the same concentration glutathione and mercaptoethanol stimulated the protease activity. These results indicate that the catabolic activity of the protease is sulfhydryl dependent. Furthermore, the activity of insulin protease was also enhanced by calcium and other divalent metal ions at a concentration of 1 mM. When supernatants, recovered from rat liver homogenates after centrifugation at 100,000g, were subjected to combined Sepharose 4B-insulin protease affinity chromatography and dialysis, a potent inhibitor of insulin protease was obtained which was heat stable. On the basis of kinetic studies, the inhibition of insulin degradation caused by this inhibitor was of the competitive type. Greater than 90% of the inhibitor activity was retained on dialysis with tubing with an inclusion limit of 3500 Da, whereas only 10% of this activity could be retained in dialysis tubing with an exclusion limit of 15,000 Da. These findings suggest that the insulin protease inhibitor is a low-molecular-weight protein. Analysis of homogenates from 13 different tissues of the rat showed that the highest levels of insulin protease inhibitor activity were associated with those tissues which have the highest capacity to degrade insulin. These data suggest that insulin protease and insulin protease inhibitor may be an important natural regulatory mechanism of insulin activity.     

Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.     

Properties of microsomal acyl coenzyme A reductase in mouse preputial glands

Synthesis of long-chain fatty alcohols in preputial glands of mice is catalyzed by an NADPH-dependent acyl coenzyme A (CoA) reductase located in microsomal membranes; sensitivity to trypsin digestion indicates that the reductase is on the cytoplasmic side of the membrane. Results with pyrazole and phenobarbital demonstrate the reaction is not catalyzed by a nonspecific alcohol dehydrogenase or an aldehyde reductase. Acyl-CoA reductase activity is sensitive to sulfhydryl and serine reagent modification, is stimulated by bovine serum albumin, and produces an aldehyde intermediate. The activity is extremely detergent sensitive and cannot be restored even after removal of the detergents. Phospholipase C or asolectin treatment does not release the acyl-CoA reductase from microsomal membranes, but causes a significant decrease in the activity recovered in the membrane pellet. Glycerol does not solubilize the reductase activity, nor does 3.0 m NaCl; however, the combination of glycerol and 3.0 m NaCl did release about 50% of the acyl-CoA reductase from the microsomal pellet. Substrate concentration curves obtained in the presence or absence of bovine serum albumin show significant differences in enzyme activities. The reductase is sensitive to the concentration of palmitoyl-CoA and is progressively inhibited at levels beyond the critical micellar concentration of the substrate. The apparent K_m for acyl-CoA reductase is 14 μm; however, the maximum velocity varies with the concentration of albumin used. Expression of enzyme activity in delipidated microsomes requires specific phospholipids, which suggests that in vivo regulation of acyl-CoA reductase activity could be achieved through modifications in membrane lipid composition.     

Regulation of acyl coenzyme A reductase by a heat-stable cytosolic protein during preputial gland development

A low-molecular-weight protein located in the cytosol of mouse preputial glands has been shown to stimulate the activity of a microsomal acyl coenzyme A (CoA) reductase in the gland. This cytoplasmic protein was stable to heating and lyophilization, but was destroyed by trypsin digestion. It was able to bind palmitoyl-CoA and gel elution behavior indicated it had a molecular weight of 10,000–12,000. The level of this stimulatory cytosolic protein and the activity of acyl-CoA reductase were shown to correlate with differentiation of the preputial gland during development of puberty in male mice; the acyl-CoA reductase activity first appeared at 4 weeks of age and increased dramatically up to 6 weeks of age. By 8 weeks, when sexual maturity was attained, the reductase activity decreased to that level found in mature male mice. The cytosol from the preputial glands of the youngest mice (3 weeks) contained sufficient heat-stable acyl-CoA binding protein to stimulate acyl-CoA reduction; however, the 3-week-old preputial gland microsomes had little or no acyl-CoA reductase activity. As the animal matured, the stimulatory capacity in the heat-treated cytosol increased, reaching a maximum at 6 weeks; by 8 weeks, the stimulatory capacity of the soluble fraction had decreased to that found in mature male mouse. Results of this study suggest that the concentration of acyl-CoA, cytoplasmic acyl-CoA binding protein, and acyl-CoA reductase activity regulate the level of fatty alcohols in vivo and that the reductase activity and binding protein have similar patterns of development during puberty.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.     

Nickel complexes of sepiapterin and 6-acetyldihydrohomopterin, a pyrimidodiazepine from Drosophila

The nonfluorescent pyrimidodiazepine in Drosophila melanogaster 6-acetyldihydrohomopterin (H₂Ahp) was studied using ultraviolet and infrared spectroscopy. The H₂Ahp was unstable in 3% NH₄Cl whereas a related pteridine sepiapterin was stable. Since Ni²⁺ stabilized H₂Ahp completely, the structure of the H₂Ahp · Ni complex was examined. Among 15 pterins, including sepiapterin, the spectral properties in the presence of Ni²⁺ reflect the pK_a's and the reactive group on the side chain but for H₂Ahp the spectral properties are rather different from the pteridines and they indicate that the seven-membered ring seemed to have the predominant influence. The Ni²⁺ comples of H₂Ahp resulted in a shift in the absorption maximum from 383 to 436 nm. The corresponding spectral shift of the pteridines due to Ni²⁺ was much less. From the infrared spectra of H₂Ahp and sepiapterin in the presence and absence of Ni²⁺, the sites of interaction of Ni²⁺ with H₂Ahp were shown to be the phenolic oxygen and N5 in the ring. In the absence of Ni²⁺ an internal hydrogen bond in sepiapterin was indicated that may involve the carbonyl oxygen and the secondary alcoholic oxygen on the side chain. Other metal ions were tested (Cd²⁺, Zn²⁺) but were not as effective as Ni²⁺ in stabilizing H₂Ahp.     

Endogenous surface phosphorylation reactions and ectokinase activity in the guinea pig T lymphocyte

The guinea pig T lymphocyte, known to interact with other cells via direct cell-to-cell contact, exhibits endogenous surface kinase activity as reflected by the appearance of four major labeled bands in autoradiographs of dried sodium dodecyl sulfate (NaDoSO4)-polyacrylamide gel electrophoresis gels when intact cells are briefly exposed to micromolar concentrations of [gamma-32P]ATP followed immediately by solubilization with NaDoSO4 to terminate the reaction. This pattern differs from the labeling of intracellular components which is seen when intact cells are incubated with 32PO4 to generate intracellular [gamma-32P]ATP when only two major labeled bands of protein with different molecular weights are seen. Of a number of modulators of lymphocyte function tested, cyclic GMP and phytohemagglutinin (PHA) caused additional bands to appear in cells exposed to [gamma-32P]ATP. The labeling of added casein was catalyzed by intact cells harvested 4 weeks after injection of animals with Freund's complete adjuvant but not earlier. These findings indicate that plasma membrane kinase activity of guinea pig T lymphocytes is accessible to the extracellular environs (ectokinase activity) and to endogenous surface substrates and that the limitation for such reactions is the availability of ATP in the extracellular component. In view of the number of circumstances under which ATP could appear outside of cells for brief periods of time, these reactions could well take place in vivo.