Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products

The inhibitory activities of c-Ha-ras gene products (p21s) toward several cysteine proteinases have been investigated. The activity of cathepsin L was inhibited by p21s most effectively while those of cathepsin B and papain were slightly inhibited by p21s. p21s did not show any inhibitory activity toward cathepsin H. In order to connect the protease-inhibitor activity of p21s with cell growth, the degradation of epidermal growth factor receptors (EGF-receptors) was investigated. EGF-receptors were preferentially cleaved by cathepsin L but not by cathepsin B or H. The cleavage of EGF-receptors by cathepsin L was inhibited by p21s dose-dependently. These results raise the possibility that p21s can suppress the degradation of growth-related proteins such as EGF-receptors and thereby affect cell growth.     

Conformational analysis and molecular dynamics simulations of maltose

Constrained conformational energy minimizations have been used to calculate an adiabatic (Φ, ψ) potential energy surface for the disaccharide β-maltose. The inclusion of molecular flexibility in the conformational energy analysis of the disaccharide was found to significantly lower the barriers to conformational transitions, as has been observed previously for other systems. Several low energy wells were identified on the adiabatic surface which differ in energy by small amounts and with low absolute barriers separating them, indicating the possibility of a non-negligible equilibrium population distribution in each well. If such a distribution of conformations existed in the physical system, the conformation observed by NMR NOE measurements would thus be a “virtual” conformation. Molecular dynamics simulations of the motions of this molecule in vacuum were also conducted and indicate that the rate of relaxation of the molecule to the adiabatic surface may be slower than the typical timescale of conformational fluctuations. This effect is apparently due to an unphysical persistence of hydrogen bond patterns in vacuum which does not occur in aqueous solution. Trajectories undergoing transitions between wells were calculated and the effects of such conformational transitions upon the ensemble mean structure, such as might be observed in an NMR experiment, were demonstrated.     

Demonstration of post-translational secretion of human placental lactogen by a mammalian in vitro translation system

This study demonstrates the post-translational translocation across the rough endoplasmic reticular membrane of a mammalian secretory protein, human preplacental lactogen. In the rabbit reticulocyte lysate, human preplacental lactogen biosynthesis is arrested by addition of cycloheximide prior to supplementation with dog pancreatic microsomal membranes, which have previously been shown to translocate and process nascent secretory proteins in a cotranslational manner. Twenty-five percent of the precursor protein is consistently converted to its mature form under these post-translational conditions. The resulting mature hormone is resistant to proteolytic degradation by added proteases, thus indicating that it is translocated across the microsomal membrane and sequestered within the lumenal space of the microsomal vesicles. Approximately one-half of the precursor protein synthesized is associated with the ribosomes. Only the ribosome-associated fraction is secreted in this in vitro system, suggesting that the process of post-translational secretion requires ribosomes for protein interaction with the elements of a subcellular secretory apparatus.     

Equilibrium binding of insulin to rat white fat cells at 15 degrees C

Equilibrium binding of insulin to isolated rat epididymal fat cells was investigated. A temperature of 15 degrees C was chosen for the study to minimize lysosomal degradation of insulin. Indeed, medium insulin lost only 1% of its precipitability in trichloroacetic acid during the 4-h incubation required to approach equilibrium. Binding was measured by a method that did not perturb the equilibrium of the system. A new formalism for analyzing binding data in general was introduced. A correction for trapping of insulin in the interstitial space of cell pellets was both necessary and sufficient to derive specific binding data from raw observations. Thus, so-called "nonspecific binding" was unmasked as a misnomer, and the expression "correction for trapping" was proposed as a substitute. Equations for one and two independent classes of binding sites were fit to the data by the method of maximum likelihood, and the best fit was selected based on Akaike's information criterion, as adapted for a constant fractional error. More than 99.7% of the binding sites were found to be describable by a simple binding isotherm with Kd,app = 8.8 multiplied by over divided by 1.3 nM. Less than 0.3% sites had a higher affinity (Kd approximately equal to 8 multiplied by over divided by 3 pM). There were 99,000 x/divided by 1.6 binding sites/cell. These equilibrium parameters are in agreement with values derived from a kinetic analysis, presented in the subsequent paper (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 260, 1702-1711).     

Native and non-native conformation-specific antibodies directed to the loop region of hen egg-white lysozyme

Two types of antibodies were differentiated in conventional guinea pig anti-hen egg-white lysozyme (HEL) antisera. The specificities of both antibodies were directed to the loop I region (mainly directed to Cys64--Cys80 loop) but the antibodies were distinct in respect of reactivities with native HEL. One type of antibody reacted with HEL and loop-peptides of HEL but not with the completely reduced and carboxymethylated form of loop-peptides (native conformation specific antibody: NC-Ab). On the other hand, the other type of antibody did not react with HEL but reacted with loop-peptides and also with the completely reduced and carboxymethylated form of loop-peptides (non-native conformation specific antibody: NNC-Ab). The percentage of NNC-Ab in loop I reactive antibody fraction from pooled guinea pig anti-HEL antisera obtained by two different immunization methods was about 25%. Since the affinities of the NNC-Ab to loop-related peptides were higher by one order of magnitude than those of the NC-Ab to the same peptides, care is necessary in evaluating antigenic determinants in native protein. The immunization of guinea pigs with Ploop I . II [sequence 57-107 (Cys64-Cys80, Cys76-Cys94)] evoked an antibody population having specificity similar to but not identical with that of the NNC-Ab type anti-loop I antibody in conventional anti-HEL antisera.     

Cytochrome b561 catalyzes transmembrane electron transfer

Purified cytochrome b561 from bovine adrenal medulla chromaffin vesicles has been reconstituted into phosphatidylcholine vesicles by a detergent-dialysis method. When the reconstituted cytochrome-containing vesicles were preloaded with ascorbic acid and cytochrome c was added to the external medium, the internal ascorbic acid was able to reduce the external cytochrome c. This reduction of cytochrome c was dependent on the presence of cytochrome b561 in the membrane and was not due to leakage of ascorbate from the vesicles. These results demonstrate that cytochrome b561 catalyzes a transmembrane electron transfer.     

Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells

Summary Chromaffin granules from bovine adrenal medullary chromaffin cells have been found to contain small vesicular structures bounded by unit membranes. Detection of these intragranular vesicles within intact cells requires the use of quick-freezing methods. The intragranular vesicles are labile to fixation by aldehydes which explains why they have not been described in intact cells until now. They are found in approximately 60% of the dense-core chromaffin granules in cells and 85% of isolated granules. They are usually clustered in groups of one to as many as five between the core and the inner surface of the granule membrane. The intragranular vesicles are independent vesicles in that they do not appear as simple invaginations of the granule membrane in either serial thin-section or freeze-etch views. Furthermore, they are released from the cell along with granule contents during nicotine-induced secretion of catecholamines. The structural heterogeneity provided by the intragranular vesicles may be related to the functional heterogeneity of granule contents observed in many recent biochemical studies.     

Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.     

Carcinoembryonic antigen content in fine needle aspirates of the lung. A diagnostic adjunct to cytology.

Carcinoembryonic Antigen (CEA) was measured in 59 consecutive fine needle aspirates (FNAs) of the lung from 58 patients to determine if the CEA content would enhance the sensitivity of the cytologic diagnosis. Twenty-eight males and 30 females with tumors 1-40 cm in diameter were studied. Final diagnoses were correlated with the clinical history, radiologic studies, tissue (when available) and follow-up. Image-guided FNAs were performed by radiologists using a 22-gauge Chiba needle and 20-mL syringe with one to four passes per specimen. Cytologic examination included rapid assessment in the radiology suite and a final diagnosis in 24 hours. CEA was measured by enzyme immunoassay using monoclonal antibody. Nine benign aspirates and 50 malignant aspirates were diagnosed. The sensitivity of cytology was 86% and specificity, 100%. Using 5 ng/mL as the cutoff, the sensitivity of CEA for malignant aspirates was 50% and specificity, 90%. The combined sensitivity of CEA and cytology was 95%. The mean CEA in malignant aspirates was 131 ng/mL and in benign aspirates, 2.41. The highest mean CEA was seen in adenocarcinoma, 402.6 ng/mL. Lower CEA content was seen in epidermoid carcinoma (58.6 ng/mL), large cell carcinoma (8.09), oat cell carcinoma, metastatic carcinoma of the kidney and breast, thymoma and lymphoma (each less than 1 ng/mL). Elevated CEA alone was diagnostic in two aspirates of bronchioloalveolar carcinoma; carcinoma with an unknown primary source, three; and large cell carcinoma, one. The adjunctive use of CEA in FNAs of the lung enhances the sensitivity of the cytologic diagnosis.