Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.     

An in vivo/in vitro evaluation of teratogenic action

Several compounds were administered to pregnant Wistar-derived rats either 24 or four hours prior to the recovery of day 10 embryos for in vitro culture in Waymouth's medium and fetal calf serum. The compounds tested were 2-amino-1,3,4-thiadiazole (thiadiazole), cadmium sulfate, 1,2-dibromo-2,2-dichloroethyl dimethyl phosphate (dibrom), 2-(sec-Butyl)-4,6-dinitrophenol (dinoseb), led nitrate, polybrominated biphenyls (PBB), sodium arsenate, and trypan blue. After 24 hours in culture, two thirds of the embryos were recovered for examination. The remaining one third were continued in culture until 42 hours. Recovered embryos were examined for rotation of the embryonic axis, heart rate, establishment of the visceral yolk sac circulation, somite number, growth of the limb buds, closure of the neural tube, and development of the allantois and amnion. All tested compounds inhibited the rate of development in vitro.     

In situ mechanical behavior of posterior spinal ligaments in the lumbar region. An in vitro study

The posterior ligaments: ligamentum flavum, articular, interspinous and supraspinous ligaments of twenty five fresh cadaveric intervertebral segments, from T11-T12 to L4-L5, extracted from fourteen spines were tested in tension. A progressive dissection method was used, that is, each segment was tested after first resecting the disk with the ligaments intact and a force-elongation curve obtained. Then one ligament was cut and the test repeated, and so on. The most restrictive ligament was found to be the ligamentum flavum followed by the articular, interspinous, and supraspinous ligaments.     

Proteolytic processing of Blattella germanica vitellin during early embryo development

In the eggs of the cockroach Blattella germanica, vitellin (Vt) utilization is initiated 4 days postovulation by the proteolytic processing of its three subunits. These reactions yield a specific set of peptides that are consumed by the developing embryo. A yolk proteinase activity, believed central to this processing event, has been investigated. First expressed at day 3 postovulation, just prior to Vt's processing, its specific activity with synthetic substrates increased four-fold to 18-fold through day 6. In addition, a mixing experiment showed that these proteinases(s) can also process Vt's large subunits in vitro. A relationship between Vt processing and proteinase specific activity was also noted with two B. germanica translocation heterozygotes, which displayed differences in the extent of Vt processing. One group of eggs (group A) failed to process any Vt subunit. A second group (B) processed the M_r 102,000 subunit but not the M_r 95,000. A third group (C) processed their Vt normally. Proteinase specific activities in the yolk of translocant's eggs at day 6 mirrored the extent of processing, being highest in group C eggs and effectively absent from the yolk of group A eggs. Eggs defective in Vt processing also contained arrested embryos. It is concluded that the yolk proteinase activity described here participates in Vt processing at day 4 postovulation. Microscopic examination of yolk obtained from eggs of wild type females showed that, as processing began in vivo (day 4), the yolk granules also underwent an abrupt decrease in size from diameters of 15–30 μm to 3–10 μm. Yolk granules of those translocant's eggs that were defective in Vt processing did not undergo this size decrease, suggesting that granule reorganization and Vt proteolysis may be linked functionally.     

Production of HMG-3 by limited trypsin digestion of purified high-mobility-group nonhistone chromatin proteins

Three isolated nonhistone proteins (HMG-1, HMG-2 and HMG-E) have been purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions, and subjected to limited proteolysis. When treated with trypsin, the three proteins exhibited similar patterns of degradation, as judged by SDS and acid/urea gel electrophoresis. In particular, the first product, P1 (a relatively stable intermediate in each digestion), was a protein analogous to HMG-3, a principal degradation product in preparations of calf thymus high-mobility-group proteins. At least in the case of HMG-E, the products formed by tryptic attack on P1 are the two individual DNA binding domains of HMG-E. P1 derived from HMG-E and one of the individual DNA binding domains of HMG-E were purified by chromatography on columns containing DNA-cellulose or phosphocellulose. The properties of these two portions of HMG-E are consistent with our recently postulated three-domain structure for HMG-1 and its homologs (Reeck, G.R., Isackson, P.J. and Teller, D.C. (1982) Nature 300, 76-78). Thus, P1 consists of two DNA-binding domains of approximately equal molecular weight covalently linked together. From chromatography on DNA-cellulose columns, it is clear that P1 binds to DNA more tightly than does HMG-E. The highly acidic C-terminal domain of HMG-E (which is removed by trypsin in generating P1) thus counteracts the DNA binding of the two other domains of HMG-E (at least in the protein's interaction with purified DNA).     

Dual action of anti-sporozoite antibodies in vitro

With the use of a double staining technique that permits localization of the sporozoite during the process of entering a host cell, we studied the biologic effects of three mAb directed against determinants contained in the circumsporozoite of Plasmodium yoelii. These mAb, which included one IgM and two IgG3, were studied in primary cultures of rodent hepatocytes inoculated with sporozoites of P. yoelii. These results confirm previous reports of the extended action of antibodies on Plasmodium falciparum after entering hepatocytes by producing a strong intrahepatocyte inhibitory effect in addition to the inhibitory effect on sporozoite entry. As with P. falciparum the intracellular effects on P. yoelii liver stages are only observed when the antibodies are present at the time the sporozoite enters the cell. While carrying out experiments on this phenomenon, it was discovered that, at lowered antibody concentrations, an increase in number of maturing liver schizonts occurs, with the increase or enhancement of infection reaching up to 150% of that of controls. It was also observed that there was an inverse relationship between the antibody concentration that was inhibitory and that which enhanced parasite infectivity.     

Secretion of gamma-glutamyltranspeptidase by the pancreas: evidence for a membrane shedding process during exocytosis

Gamma Glutamyltranspeptidase (GGT) is a membrane-bound enzyme involved in glutathione metabolism. It is present in rat exocrine pancreas at a level which is only exceeded by the kidney. It has been previously shown that most of the enzyme activity is located in the apical area of the acinar cell, more precisely at the level of zymogen granules and plasma membrane. The aim of the present study was to examine the secretory behavior of that enzyme. Under resting conditions, in vivo, high levels of GGT were found in pancreatic juice and its level was not related to protein concentration. Under secretin infusion, a relatively constant level of GGT was released, and again, there was no correlation between enzyme activity and protein concentration. Following a bolus injection of caerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. This observation is in agreement with in vitro determinations on isolated zymogen granules showing that GGT is totally associated with the ZG membrane and undetect-able in the content of these organelles. The present data show that 1 degree GGT is released from the rat pancreas acinar cells in a particulate form; 2 degree GGT release is elicited by hormonal stimulation coinciding with the exocytotic release of secretory proteins. Our observations lead us to propose that in rat pancreas, ZG membrane fragments are released along with secretory proteins during exocytosis.     

Reversible inhibition of proton release activity and the anesthetic-induced acid-base equilibrium between the 480 and 570 nm forms of bacteriorhodopsin.

In purple membrane added with general anesthetics, there exists an acid-base equilibrium between two spectral forms of the pigment: bR570 and bR480 (apparent pKa = 7.3). As the purple 570 nm bacteriorhodopsin is reversibly transformed into its red 480 nm form, the proton pumping capability of the pigment reversibly decreases, as indicated by transient proton release measurements and proton translocation action spectra of mixture of both spectral forms. It happens in spite of a complete photochemical activity in bR480 that is mostly characterized by fast deprotonation and slow reprotonation steps and which, under continuous illumination, bleaches with a yield comparable to that of bR570. This modified photochemical activity has a correlated specific photoelectrical counterpart: a faster proton extrusion current and a slower reprotonation current. The relative areas of all photocurrent phases are reduced in bR480, most likely because its photochemistry is accompanied by charge movements for shorter distances than in the native pigment, reflecting a reversible inhibition of the pumping activity.     

Changes in S-type lectin localization in neuroblastoma cells (N1E115) upon differentiation

The distribution of a 14.4 kDa S-type lectin was examined in murine neuroblastoma cells, either undifferentiated or after differentiation induced by dibutyryl-cyclic adenosine monophosphate. In undifferentiated cells the immunoreactivity was detected extracellularly, associated with the plasma membrane and in bulges released into the extracellular milieu. Important modifications of the lectin localization were associated with the differentiation process that induced an increased cytosolic expression and a decreased externalization. Possible functions for the lectin expressed intracellularly in the differentiated cells are also considered.