Purification and properties of human intestine alanine aminopeptidase

Human intestinal alanine aminopeptidase has been purified to greater than 90% homogeneity. The enzyme was released from mucosal cell membranes by Triton X-100 treatment. The native enzyme had a molecular weight of 206,000 in dilute buffer and 108,000 in the presence of sodium dodecyl sulfate. The enzyme was inhibited by chelators suggesting the presence of a metal ion in the enzyme. The most potent chelator inhibitor tested, o-phenanthroline, gave mixed kinetics (Ki = 67 micro M). Activity was restored by removal of the chelator. The enzyme was inhibited competitively by amino acids having hydrophobic side chains such as L-phenylalanine (Ki = 0.67 mM). Puromycin and methicillin also inhibited the enzyme in the competitive (Ki = 12.5 micro M) and noncompetitive (Ki = 4.6 mM) manner, respectively. Kinetic analysis of several amino acid beta-naphthylamides as substrates demonstrated the preference for substrates having hydrophobic or basic amino terminal residues with no beta-branching. L-Methionyl-beta-naphthylamide was the most tightly bound with L-alanyl-beta-naphthylamide was the most rapidly hydrolyzed.     

Ultrastructural analysis of the granulosa — Luteal cell transition in the ovary of the dog

The transition from ovarian granulosa to lutein cell during the estrus cycle of 60 pregnant and non-pregnant beagle bitches was analyzed by light and electron microscopy (both 100 and 1000 KV). Early proestrus was characterized by a gradual rise in serum estrogen levels, hyperplasia of the granulosa cells, the accumulation of follicular fluid, and the development of tortuous intercellular channels. During the second half of proestrus, serum estrogen levels continued to rise, but growth, division, and differentiation of the granulosa cells was minimal. Estrus was marked by the first acceptance of the male and a well-defined LH peak In the subsequent 24 hour period, the granulosa-lutein cells hypertrophy rapidly and develop a large Golgi apparatus, small profiles of granular endoplasmic reticulum, numerous microfilaments, and large gap junctions between the cells. Mitochondria also proliferate, enlarge, and elongate, but retain lamelliform cristae. Luteinization of the cells and progesterone secretion begin just after ovulation which in turn occurs about 24 hours after the LH peak. On the third and fourth day of estrus, numerous small vesicles of agranular endoplasmic reticulum fill the extoplasm and the mitochondria swell up and round off. The vesicles rapidly fuse into whorled and flattened cisternae or anastomosing tubules of agranular endoplasmic reticulum, while the mitochondria develop tubulovesicular cristae. These structures gradually become organized with respect to the basal lamina. The Golgi apparatus is centered over the pole of the nucleus that faces the pericapillary space. Stacked and whorled cisternae of agranular ER develop in the lateral margins and avascular end of the cell while mitochondria and tubular elements of agranular ER predominate in the central medial and most basal portions of the cytoplasm. Microfilaments are ubiquitous and appear to be instrumental in this orientation process. The cell surface develops three distinct regional specializations that coincide with the underlying cellular compartments: interconnecting pleomorphic folds fill the pericapillary space; long tenous microvilli project from the lateral cell surface and form tortuous intercellular channels and canaliculi; and large gap junctions form along the margins of the cell furthest removed from the basal lamina. By the sixth day of estrus, the granulosa-luteal cell transition is nearly complete and serum progesterone levels are on the rise.     

Trophic interactions in soils as they affect energy and nutrient dynamics. III. Biotic interactions of bacteria,amoebae, and nematodes

Bacteria (Pseudomonas), amoebae (Acanthamoeba), and nematodes (Mesodiplogaster) were raised in soil microcosms with and without glucose additions. Nematode and amoebal grazing on bacteria significantly reduced bacterial populations by the end of a 24-day incubation period. Amoebal numbers decreased in the presence of nematodes with a corresponding increase in nematode numbers which reached a maximum of 230 nematodes/g of soil in the treatment with amoebae and glucose additions. After 24 days the nematode populations in the treatments without carbon additions were dominated by resistant dauer larvae indicating the unavailability of food. Although larval numbers were high in the treatments with glucose additions, the adult component of the population was still increasing at the end of the 24-day experiment. The effect of the presence of amoebae on nematode abundance was of the same magnitude as addition of 600g glucose-C.     

Electron probe X-ray microanalysis of post-tetanic Ca2+ and Mg2+ movements across the sarcoplasmic reticulum in situ

Ca2+ and Mg2+ movements across the sarcoplasmic reticulum (SR) of frog skeletal muscle fibers were measured in situ by electron probe microanalysis of muscles rapidly frozen following a tetanus. At 400 ms following a 1.2-s tetanus at room temperature, the force had relaxed to base-line, and 0.3 mmol of Ca2+/liter of cytoplasmic H2O had been pumped by the SR, indicating that the in situ pumping of the SR Ca-ATPase is sufficiently high to account for the removal of Ca2+ from the Ca2+-specific sites of troponin (0.18 mmol of Ca2+-specific sites/liter of cytoplasmic H2O) and for the rate of relaxation from a tetanus at room temperature. The half-time of the return of the total 1.0 mmol of Ca2+/liter of cytoplasmic H2O released during a tetanus was 1.1 s, comparable to the slow Koff rate of Ca2+ from (carp) parvalbumin (1.0 s-1) and consistent with the hypothesis that the return of this Ca2+ to the terminal cisternae is rate-limited by the Ca2+ off-rate from parvalbumin. The return of the Mg2+ taken up by the terminal cisternae during a tetanus to resting levels was significantly slower than the time course of the Ca2+ movements, suggesting that the Mg2+ permeability of the SR in situ is low and may be transiently increased during tetanic stimulation.     

Interaction of the retinal insulin receptor beta-subunit with the p85 subunit of phosphoinositide 3-kinase

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor beta-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRbeta) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta. Under conditions where IRbeta autophosphorylates, substitution of Y1322F and M1325P in IRbeta resulted in the abolition of p85 binding to the IRbeta, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRbeta was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRbeta (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRbeta-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable of bringing down PI3K activity from retina lysates. On the other hand, in response to insulin, IRS-2 is able to interact with the p85 subunit of PI3K in the retina. These results suggest that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.     

Naphthamidine urokinase plasminogen activator inhibitors with improved pharmacokinetic properties

A series of non-amide-linked 6-substituted-2-naphthamidine urokinase plasminogen activator (uPA) inhibitors are described. These compounds possess excellent binding activities and selectivities with significantly improved pharmacokinetic profiles versus previously described amide-linked inhibitors.