Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.     

Immunochemical study on PHI/PHM with use of synthetic peptides

We have synthesized PHI and PHM (human PHI) as well as their fragments, PHI (1-6), PHI (1-15), PHI (14-19), PHI (14-27), PHI (20-27), PHM (1-15) and PHM (13-27), by the solution or solid-phase method for peptide synthesis. Using the highly purified synthetic peptides as immunogens or haptenic immunogens, five kinds of PHI/PHM specific antisera were produced. The major antibody-recognition sites of the five antisera were located respectively in the PHI C-terminal (R8201), in the PHI N-terminal (R8403), in the PHM C-terminal (R8502), and in the PHM whole molecule (R8702 and R8703). Radioimmunoassays (RIAs) with antisera R8201, R8403 and R8502, respectively, showed a wide distribution of immunoreactive (IR) PHI/PHM in porcine and human gastrointestinal and brain tissues. The concentrations of IR-PHI in the porcine gastrointestinal tissues, however, differed between the R8201 and R8403 RIAs employed for measurement. By using these two different PHI RIAs, the IR-PHI in the porcine brain tissue extract was shown to be almost a single component coeluting with synthetic PHI in gel filtration. The IR-PHI in the extract of porcine lower intestine on the other hand, contained, besides a PHI-like component, unidentified component(s) eluting immediately after synthetic PHI in gel filtration; this crossreacted with the PHI C-terminal specific R8201 antiserum but not with the N-terminal specific R8403 antiserum, suggesting the presence of the C-terminal-related fragment(s) of PHI in the tissues.     

Radioimmunoassay of neuropeptide Y

The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences.     

Existence of a common precursor to ACTH and endorphin in the anterior and intermediate lobes of the rat pituaitary

Extracts of rat anterior and intermediate-posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for immunoactive ACTH and endorphin. In both lobes the major forms of immunoactive ACTH have apparent molecular weights of 31,000 (31K), 20–21K, 14K, and 4.5K, and the major forms of immunoactive endorphin have apparent molecular weights of 31K (coincident with the peak of immunoactive ACTH), 13K (a βLPH-like peptide), and 3.5K (a β-endorphin-like peptide). However, the quantitative distribution of immunoactivity among the various forms differs greatly between the lobes. Assays using an extreme COOH-terminal ACTH antiserum indicate that the 31K ACTH/endorphin molecule in rat antierior and intermediate pituitary is similar to the pro-ACTH/endorphin molecule from mouse pituitary tumor cells. A radioimmunoassay that is specific for the NH₂-terminal non-ACTH, nonendorphin segment (referred to as 16K fragment) of the mouse pro-ACTH/endorphin molecule was used to assay extracts of rat pituitary. In addition to detecting material at 31K and 20–21K, the 16K fragment radioimmunoassay detects significant amounts of cross-reactive material with an apparent molecular weight of 16K in extracts of both lobes. This result also suggests that the structure and processing of the rat 31K ACTH/endorphin molecule is similar to that of mouse tumor cell pro-ACTH/endorphin. Cell suspensions were prepared from the anterior and intermediate lobes of the rat pituitary and maintained in culture for a 24-h period. The isolated cells from both lobes incorporate [³H] phenylalanine into immunoprecipitable ACTH- and endorphin-containing molecules. By sequential immunoprecipitation with ACTH and endorphin antisera, it is possible to demonstrate directly that a single molecule (31K ACTH/endorphin) has antigenic determinants for both ACTH and endorphin. Significant amounts of 31K ACTH/endorphin are released into the culture medium by isolated anterior lobe and intermediate lobe cells. The isolated intermediate lobe cells synthesize and secrete relatively large amounts of a β-endorphin-like molecule; the isolated anterior lobe cells secrete significant amounts of both a βLPH-like molecule and a β-endorphin like molecule. These same quantitative differences between anterior and intermediate lobe tissue were observed in immunoassays of extracts of the separated lobes and probably reflect differences in the processing of the common precursor. The isolated anterior lobe cells can be stimulated to release increased amounts of immunoprecipitable ACTH and endorphin by incubation with a cyclic AMP analog and a phosphodiesterase inhibitor.     

COMPARISON OF ATTACHED DIATOM COMMUNITIES ON NATURAL AND ARTIFICIAL SUBSTRATES1

A study of attached diatom communities on artificial and natural substrates was conducted in Wheelwright Pond, New Hampshire, during 1975. There were differences in the species composition growing on artificial glass slides and natural substrates. The slides favored the accumulation of Achnanthes minutissima Kütz. and decreased the relative abundance of Eunotia incisa W. Sm. ex. Greg. and Cocconeis placentula v. euglypta (Ehr.) Cleve. Large growths of planktonic species were also noted on the slides. Compositional differences between slides positioned at 15–30 cm (upper) and 1 m (lower) from the surface of the water were minimal. A similar community composition of periphytic diatoms was found on five aquatic macrophytes.     

The binding of cytochrome b5 to plasma membranes of rat liver. Its implication for membrane specificity and biogenesis

The in vitro incorporation of cytochrome $\text{b}{}_5$ into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome $\text{b}{}_5$ than did microsomal preparations; 60% of this cytochrome $\text{b}{}_5$ could not be reduced by the NADH-cytochrome $\text{b}{}_5$ reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome $\text{b}{}_5$ clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome $\text{b}{}_5$. These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell.     

On the specificity of the uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: Diamino acid ligase of Bifidobacterium globosum

The peptidoglycan of Bifidobacterium globosum contains ornithine and lysine alternately in the same position of the peptide subunit. The uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: diamino acid ligase of this organism was purified 700-fold. Since the activities for the incorporation of ornithine and lysine into uridine diphospho-N-acetylmuramyl-tripeptide did not separate during purification and since the incorporation of ornithine is competitively inhibited by lysine and vice versa, both ornithine and lysine are assumed to be incorporated by one single enzyme. Studies on the specificity of the ligase toward analogs of ornithine have shown that the enzyme requires a diamino, monocarboxylic acid with 4–6 carbon atoms. Methylation of the -amino group or hydroxylation of the -carbon atom of lysine decreases the competitive properties of the analog, whereas the substitution of the -methylen group by sulfur (S-2-aminoethyl cysteine) results in a highly competitive compound.Abbreviations BSA bovine serum albumine - MurNAc N-acetyl-muramyl - DA diamino acid - Ala-DGlu--L-DA-DAla-D-Ala pentapeptide - Ala-DGlu--LDA tripeptide - Ala-DGlu dipeptide - DSM Deutsche Sammlung von Mikroorganismen - CEM clostridial enrichment medium     

Aggregative properties of different cell types of the fresh-water spongeEphydatia fluviatilis isolated on ficoll gradients

Summary Cell suspensions of the fresh-watersponge Ephydatia fluviatilis have been fractionated by means ofFicoll gradient centrifugation. Three fractions were isolated. The densest contains archeocyte-like cells only; the intermediate fraction is very rich in choanocytes, and the lightest is a mixture of cell types. Earch fraction shows specificaggregative properties and potentialities to reconstitute functional sponges.It appears that the sequence of reconstitution events can be selectively altered by certain disequilibria in the cell populationThese preliminary results constitute a first approach to the analysis ofcell type specificity in sponges.     

High Expression of the HNK-1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.     

Cardiodynamic response to psychological and cold pressor stress: Further evidence for stimulus response specificity and directional fractionation

In the present study 36 police officers were exposed to a psychological stressor (IQ quiz) and to cold pressor stress while several cardiovascular variables were monitored. Impedance cardiography was used to provide measures of heart rate, stroke volume, cardiac output, myocardial contractility, and total peripheral resistance. In addition, measures of systolic and diastolic blood pressure and peripheral skin temperature were obtained. A multivariate analysis of variance (MANOVA) indicated that significant increases in diastolic and systolic blood pressure during the cold pressor test were mediated by large increases in total peripheral resistance, whereas blood pressure elevation during the IQ quiz were accompanied by significant increases in heart rate and, to a lesser extent, cardiac output. Peripheral skin temperature decreased in response to each stressor. Additional analysis indicated a degree of stimulus specificity for several variables. For example, diastolic blood pressure showed greater increases to cold pressor than quiz, whereas systolic blood pressure increased more with the psychological than the physical stressor. Directional fractionation occurred for both myocardial contractility and cardiac output.