Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase,legumain

To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a “fragment assay” of autophagic activity in whole cells.

Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease.     

Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain

To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a "fragment assay" of autophagic activity in whole cells. Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease.     

Macroautophagy-dependent, intralysosomal cleavage of a betaine homocysteine methyltransferase fusion protein requires stable multimerization

Cargo-based assays have proven invaluable in the study of macroautophagy in yeast and mammalian cells. Proteomic analysis of autolysosomes identified the metabolic enzyme, betaine homocysteine methyltransferase (BHMT), as a potential cargo-based, end-point marker for mammalian macroautophagy. To test whether degradation of BHMT can be used to measure macroautophagic flux in mammalian cells, we created a BHMT fusion protein (GST-BHMT) that demonstrates starvation-induced, site-specific fragmentation in a variety of cell lines. Subcellular fractionation studies show that the GST-BHMT fragment co-fractionates with vesicles containing lysosomal and autolysosomal markers. Furthermore, both pharmacological inhibitors of macroautophagy and depletion of macroautophagy-specific proteins reduce accumulation of the fragment. In the course of these studies, we observed that fragmentation of GST-BHMT did not occur in forms of the reporter with truncation or point mutations that destabilize oligomerization. Since stable oligomerization of BHMT is essential for its catalytic activity, a point mutation known to ablate BHMT activity was tested. We show that accumulation of the GST-BHMT fragment is not impaired in a catalytically inactive mutant, indicating that selective proteolysis of GST-BHMT requires stable quaternary structure independent of effects on activity. Also, the loss of fragmentation observed in the oligomerization deficient mutants does not seem to be due to a defect of sequestration and lysosomal loading, suggesting that disruption of stable quaternary structure affects the ability of a lysosomal protease to cleave the newly-delivered cargo. Finally, we propose that the cargo-based GST-BHMT assay will be a valuable addition to existing macroautophagy assays in mammalian cells.     

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

In vitro activation of neuraminidase in the beta-galactosidase-neuraminidase-protective protein complex by cathepsin C

Neuraminidase can be activated by incubation of crude glycoprotein fractions at acidic pH for 90 minutes at physiological temperature. This activation is inhibited by leupeptin. Incubation of the purified neuraminidase-beta-galactosidase-protective protein complex under the same conditions used for crude glycoprotein fractions did not lead to enhanced neuraminidase activity, but incubation in the presence of exogenous Cathepsin C at 4 degrees C resulted in marked enhancement of neuraminidase activity. This activation was again inhibited by leupeptin. Cathepsin D treatment resulted in destruction of neuraminidase under the same conditions and this effect was again inhibited by leupeptin. beta-galactosidase in crude glycoprotein fractions and in the complex was resistant to both Cathepsin C and D, while homogeneous beta-galactosidase was inactivated by these enzymes. We suggest that in vitro activation of neuraminidase may mimic the in vivo intralysosomal conversion of the neuraminidase precursor into the mature form of the enzyme.     

Processing of V-ATPase subunit B of Mesembryanthemum crystallinum L. is mediated in vitro by a protease and/or reactive oxygen species

Soluble proteins were isolated from leaves of the common ice plant Mesembryanthemum crystallinum L. in the CAM state of photosynthesis and tested for protease activity using amino acid-beta-naphthylamide (NA)-derivatives in a search for proteolytic activity responsible for cleavage of the V-ATPase subunit B. This cleavage is suggested to occur at the peptide bond between Met192 and Glu193. At neutral pH Met-NA was one of seven derivatives which were cleaved by proteases present in this fraction. Enzymes exhibiting proteolytic activity were separated from other soluble proteins by Superose 12-size exclusion FPLC. Incubation of partially purified protease with tonoplast-enriched membrane vesicle fractions isolated from M. crystallinum in the C3-state of photosynthesis led to a decrease in subunit B (55 kDa) protein amount and to the formation of the polypeptide Di (32 kDa), which has been previously suggested to represent a fragment of subunit B. Cleavage of subunit B and the appearance of Di also occurred during incubation of tonoplast vesicles in the presence of reactive oxygen species. In addition to Di, the polypeptide Ei (28 kDa) appeared after incubation with protease and/or reactive oxygen species. Taken into account that Di and Ei cross-reacted with an affinity purified antiserum directed against subunit B, Di as well as Ei might represent fragments of subunit B. These results open new perspectives with respect to the regulation of V-ATPase modification and turnover.     

Characterization of macroautophagic flux in vivo using a leupeptin-based assay

Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.     

Inhibition of glycoprotein catabolism in vivo and in the perfused rat liver.

Leupeptin is a peptide which inhibits several of the lysosomal proteases. When this compound was added in low concentrations to a perfused liver, the degradation of 125I-asialo-fetuin by the liver was dramatically slowed. When 5 mg leupeptin were added to the perfusate 1 h prior to the radioactive glycoprotein, the liver retained from 70 to 90% or the radioisotope 60 min after infusing 125I-asialo-fetuin. However, untreated livers contained less than 20% of the radioactivity at that time. Subcellular fractionation experiments showed that the radioactivity accumulated in the heavy and light mitochondrial fractions (ML) of the homogenate. At 80 min after the glycoprotein was added, almost 40% of the radioactivity was still located with these fractions. Very similar inhibitory effects were seen upon treating rats intravenously with 5 mg of leupeptin 60 min prior to injection of 125I-labelled asialo-fetuin. A 7 fold increase in liver radioactivity was observed 6 hrs after the glycoprotein had been given to the treated animals. Purified human liver cathepsin B digested fetuin to about 3% of total hydrolysis and the major peptide fragment produced had an SDS-electrophoretic mobility equivalent to that of ovalbumin.     

Insulin processing and action in adipocytes: evidence for generation of insulin-containing vesicles by leupeptin and monensin

Incubation of adipocytes with 125I-insulin plus leupeptin or monensin, but not chloroquine, resulted in the appearance of a novel peak of 125I-insulin (modal density about 1.20 g/ml) on density gradient centrifugation; the appearance of the peak depended on the presence of specific insulin receptors on the cell surface. The fractions comprising this peak contained vesicles, probably originating from the Golgi apparatus, and dit not appear to be contaminated with lysosomes, mitochondria or plasma membrane. Entrapment of insulin in these vesicles per se did not prevent the activation of glucose transport, acetyl-CoA carboxylase or pyruvate dehydrogenase by insulin.     

Characterization of macroautophagic flux in vivo using a leupeptin-based assay

Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.     

Protein p126: a parasitophorous vacuole antigen associated with the release of Plasmodium falciparum merozoites

The p126 protein is synthesized by P. falciparum between the 32nd and the 36th hour of the erythrocytic cycle, and is localized in the parasitophorous vacuole. It is processed when schizonts rupture and the major fragments (50, 47 and 18 kDa), which are released into culture supernatant, have been characterized using monoclonal antibodies. The 47 kDa fragment has been mapped at the N-terminus of the molecule. The portion of the protein p126 gene coding for this fragment contains 3 introns and is characterized by a sequence coding for 6 repeats of 8 aminoacids and by repeats of TCA/T-AGT coding for a polyserine sequence of 37 serines in a row for the FCR-3 strain. The 50 kDa fragment is also found in culture supernatant when merozoites are released from mature schizonts. The incubation of mature schizonts with leupeptin inhibits the release of merozoites and, in this case, a 56 kDa intermediate product is found. In those conditions, merozoites were observed free in the erythrocyte cytoplasm, the membrane of the parasitophorous vacuole being destroyed. The 50 kDa fragment can be obtained from the 56 kDa fragment by treatment with trypsin (a protease inhibited by leupeptin). Our results suggest that the processing of the 56 kDa fragment: 1) is protease-dependent, and could depend on a trypsin-like activity; 2) cannot occur after the release of merozoites because of the protease inhibitors contained in the serum; 3) does not occur before the release of merozoites, since no processed products of the protein p126 are observed in unruptured schizonts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin

Receptor-mediated endocytosis of 125I-insulin and 125I-prolactin into liver parenchymal cells has been studied by quantitative subcellular fractionation. Differential centrifugation yielded three particulate fractions, N (nuclear), ML (large granule), and P (microsomes), and a final supernatant (S). Quantitative differences in the extent and rates of accumulation of 125I-insulin and 125I-prolactin into the fractions were observed. The acidotropic agent chloroquine and the microtubule disrupting agent colchicine were administered separately to rats. The agents increased significantly the T 1/2 of hormone clearance from the liver and augmented the accumulation of both ligands in the low-speed ML fraction. However, differences in the rates of accumulation of insulin and prolactin into all cell fractions were still maintained. Analytical centrifugation of each of the particulate fractions was carried out in order to determine if different endocytic components were specific to insulin or prolactin internalization. This was not the case. An "early" endosomal component of density 1.11 was identified in microsomes. A "late" endosome of density 1.10 was identified in the large granule (ML) fraction. Both endosomal components appeared to accumulate insulin and prolactin but at different rates. Marker enzyme analysis identified the presumed plasma membrane component in microsomes (density approximately 1.155). This component showed a significant difference in the rate of loss of 125I-insulin (T 1/2 approximately 4.1 min) as compared to that of 125I-prolactin (T 1/2 approximately 12.7 min). A further difference in the handling of the ligands was observed in early endosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled iodoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3′,5′-(cyclic)-adenosine monophosphate

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Chain-shortening of prostaglandin F2 alpha by rat liver peroxisomes

Liver peroxisomes were isolated from di(2-ethylhexyl)phthalate treated rats by isopycnic sucrose gradient centrifugation of a light mitochondrial fraction. Incubation of prostaglandin F2 alpha with purified peroxisomes resulted in conversion into a more polar product(s). In contrast, incubation with mitochondrial fractions and microsomal fractions under the same conditions did not result in any detectable conversion. The polar material obtained from a preparative incubation was purified by high performance liquid chromatography and characterized by radio-gas chromatography and gas chromatography-mass spectrometry. The structure of the polar compound was shown to be 5,7,11-trihydroxy-tetranorprost-9-enoic acid (tetranor-prostaglandin F1 alpha). Prostaglandin F2 alpha was thus chain-shortened by four carbon atoms.     

The inhibition of a chromatin-bound proteolytic activity

A chromatin-bound proteolytic activity found in rabbit thymus nuclear lysates has been studied. Incubation of washed chromatin fibers led to degradation of histone H1 resulting in products which ran on sodium dodecyl sulfate-gels slightly ahead of the three native H1 fractions. The activity has also been demonstrated in a chromatin preparation purified by chromatography on Sephadex G-200. Of the five histones, H1 appeared to be the most readily degraded although with extended incubation times the core histones were also affected. The proteolytic activity was not inhibited by Na₂SO₃, PMSF, STI, pepstatin, or EDTA. Effective inhibitors included leupeptin, tosyllysine chloromethyl ketone, and tosylaminophenylethylchloromethyl ketone.     

Morphological changes in cultured myotubes treated with agents that interfere with lysosomal function

Summary Treatment of cultured muscle cells with the inhibitors of lysosomal function, leupeptin, and chloroquine, decrease the degradation of acetylcholine receptors (AChR) and causes accumulation of undegraded receptors intracellularly. Under these conditions the number of cytoplasmic coated vesicles, i.e. structures that appear to transport this receptor within the cultured muscle cell, increases in parallel. This study investigates the effects of leupeptin and chloroquine on the morphology of cultured myotubes in order to learn more about the turnover of acetylcholine (ACh) receptors and the origin of the coated vesicles. Chloroquine causes involution of the plasma membrane, disorganization in the arrangement of sarcomeres, vacuolization, and enlargement of dense lysosome-like bodies in myotubes. The diameter of dense bodies in untreated myotubes is 0.36 ± 0.01 m (mean ± SEM) compared with 2 ± 0.12 m after 48 h of incubation with chloroquine. Leupeptin does not disrupt the normal architecture of sarcomeres and does not cause vacuolization of the myotubes. However, leupeptin does enlarge the dense bodies, although to a lesser extent than chloroquine (average diameter after 48 h treatment, 1.0 ± 0.06 m, p < 0.01). Untreated myotubes appear to contain equal numbers of large and small coated vesicles. After chloroquine treatment 95% of coated vesicles are large (80–120 nm in diameter), whereas after leupeptin treatment the majority of coated vesicles are small (40–70 nm in diameter). After incubation with horseradish peroxidase (HRP) 62% ± 9 of coated vesicles in chloroquinetreated cells contain the tracer, whereas in control cells only 11% ± 4 of coated vesicles contain HRP reaction product. These observations indicate that chloroquine causes accumulation of coated vesicles and interferes with degradation of AChR by preventing fusion of lysosomes with coated vesicles originating by endocytosis.     

Uptake of oestradiol by the rabbit hypothalamus. Specificity of binding by nuclei in vitro

The binding of [6,7-(3)H]oestradiol-17beta to subcellular fractions of the hypothalamus and the cerebellum of the rabbit was studied in vitro. Uptake of steroid was higher in hypothalamic nuclei than in cerebellar nuclei. Lower binding was observed in other fractions of both tissues. After dialysis of the fractions, hypothalamic nuclei retained a high percentage of oestradiol whereas cerebellar nuclei lost most of the bound steroid. Supernatant fractions of both tissues retained a significant proportion of label after dialysis and after gel filtration on Sephadex G-200. No specific binding was observed in these fractions when subjected to sucrose-density-gradient centrifugation. Purification of nuclei followed by incubation with labelled oestradiol in the absence of the supernatant fraction resulted in loss of binding of steroid by hypothalamic nuclei. Incubation of the purified hypothalamic nuclei with supernatant fraction maintained the binding specificity of hormone retention.     

Glycoprotein Ib in the triton-insoluble (cytoskeletal) fraction of blood platelets

Glycoprotein Ib could be demonstrated in the Triton-insoluble (cytoskeletal) fraction of platelets prepared with EGTA by SDS-polyacrylamide gel electrophoresis and staining with the periodic acid Schiff's reagent. Crossed immunoelectrophoresis showed that glycoprotein Ib could be extracted from such Triton-insoluble residues when the extraction solution contained 1% Triton X-100 plus 5 mM CaCl₂, but not if it also contained leupeptin. This indicates that glycoprotein Ib was associated to structures in the cytoskeletal fraction in such a way that it could be extracted only after activation of a calcium-dependent protease, and degradation of the actin-binding protein was demonstrated. After crossed immunoelectrophoresis of platelet extracts prepared in the presence of leupeptin or EDTA, a glycoprotein Ib-related, rocket-shaped immunoprecipitate was seen originating from the application well. This was interpreted as being related to glycoprotein Ib associated to actin polymers which did not sediment at low-speed centrifugation. Incubation of platelets with ³²P as sodium phosphate led to incorporation of phosphatase-sensitive ³²P in all of the glycoprotein Ib-related immunoprecipitates except for that of glycocalicin. This support the idea that glycoprotein Ib traverses the plasma membrane and can be phosphorylated at the inner surface whereas glycocalicin represents the terminal part of the glycoprotein Ibα-chain exposed at the outer surface.     

Possible MIS Production by Follicle Cells in Spontaneous Oocyte Maturation of the Ascidian, Halocynthia roretzi

Outer and inner follicle cell-enclosed oocytes (oocyte complexes) of Halocynthia roretzi underwent germinal vesicle breakdown (GVBD) within 2 hr when transferred from ovaries to normal seawater of pH 8 (NSW). Extrusion of test cells (TC) into the perivitelline space and elevation of the chorion also occurred. This phenomenon was designated as spontaneous oocyte maturation.
Seawater of low pH, protease inhibitors such as leupeptin or soybean trypsin inhibitor (SBTI), and calcium deficiency inhibited the spontaneous maturation only when introduced to the NSW during the first 10 minutes of incubation. GVBD-blocked complexes underwent GVBD after addition of trypsin regardless of pH or the absence of calcium ions. The oocytes from which follicle cells were removed with glycosidase did not undergo GVBD in NSW, but addition of trypsin triggered GVBD in these defolliculated oocytes (TC oocytes). Furthermore, incubation media in which spontaneous maturation had occurred, induced GVBD in the TC oocytes. This GVBD-inducing activity was heat-labile and was inhibited by leupeptin.
These results indicate that in the first step of the spontaneous oocyte maturation, outer and/or inner follicle cells give a signal to the oocyte itself or TC oocyte. This signal is likely to be trypsin-like.