Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.     

Structural basis of substrate specificity in the serine proteases.

Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system.     

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.     

Renin cleavage of a human kidney renin substrate analogous to human angiotensinogen, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, that is human renin specific and is resistant to cathepsin D

A synthetic tetradecapeptide, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, which corresponds to the 13 amino terminal residues of human angiotensinogen plus a carboxy terminal serine to replace a suggested site of carbohydrate attachment, has been shown to be a good substrate for human kidney renin. At pH 7.2 and 37 degrees C the KM or Michaelis constant was 8.4 +/- 2.9 microM, and the VM or velocity at infinite tetradecapeptide concentration was 11.3 +/- 2.4 mumol angiotensin I made per hour per milligram renin. The tetradecapeptide was highly resistant to cleavage by mouse submaxillary renin. The tetradecapeptide was also slowly cleaved by human liver cathepsin D, by rabbit lung angiotensin-converting enzyme, and by reconstituted human serum, but did not yield angiotensin I. Thus, this synthetic renin substrate should permit more specific measurement of human kidney renin activity.     

Size-related selection of food plants by bumblebees

Abstract. 1. A positive correlation between the tongue length of conspecific workers collecting nectar from seven plant species and the corolla length of the flowers probed was found for B.lapidarius and B.pascuomm but not B.terrestris . No simple relationship was found between the volume, sugar weight or concentration of nectar in flowers and the tongue or wing length of probing bees.
2. B.terrestris workers collecting pollen from four plant species producing pollen only, were found to differ in size according to the type of pollen presentation mechanism and the pollen content per flower. Body size variation was also related to the foraging of pollen plus nectar from two other plant species.     

Influence of culture parameters on lignin metabolism byPhanerochaete chrysosporium

Culture parameters influencing metabolism of synthetic¹⁴C-lignins to¹⁴CO₂ in defined media have been studied in shallow batch cultures of the ligninolytic wood-destroying HymenomycetePhanerochaete chrysosporium Burds. Study of the effect of O₂ concentration in the gas phase above non-agitated cultures indicated essentially complete absence of attack on the lignin polymer at 5% O₂ in N₂, and a 2- to 3-fold enhancement by 100% O₂ as compared to air (21% O₂). Agitation of the cultures resulting in the formation of mycelial pellets greatly suppressed lignin decomposition. The optimum culture pH for lignin decomposition was 4 to 4.5, with marked suppression above 5.5 and below 3.5. The source of nutrient nitrogen (NO ₃ ^– , NH ₄ ⁺ , amino acids) had little influence on lignin decomposition, but the concentration of nitrogen was critical; decomposition at 24 mM was only 25–35% of that at 2.4 mM N. Thiamine was the only vitamin required for growth and lignin decomposition. Under the optimum conditions developed, decomposition of 5 mg of synthetic lignin was accompanied by utilization of approximately 100 mg of glucose. The influence of the various culture parameters was analogous for metabolism of synthetic lignin labeled in the ring-,side chain-, and methoxyl carbon atoms.     

Correction of severe manganese deficiency in wheat with chemical fertilizers

Summary Manganese, N and P fertilizers were applied to wheat in field experiments on a soil so deficient in Mn that it caused the wheat to die before heading. Yields of wheat were increased linearly by soil banded Mn to 44.8 kg/ha, giving a yield of 3.03 tonnes/ha. Yields were increased to a lesser extent by foliar-applied Mn and least by soil-broadcasted Mn. Soil N and P appeared to be adequate, yet ammonium sulphate at 56 kg N/ha where applied alone caused a yield of 1.69 tonnes/ha and ammonium sulphate nitrate gave a yield of 0.98 tonnes/ha, the increases being primarily due to the release of Mn to the plants. Calcium nitrate and triple superphosphate were much less effective in releasing Mn.     

STUDIES OF HAWAIIAN FRESHWATER AND SOIL ALGAE II. ALGAL COLONIZATION AND SUCCESSION ON A DATED VOLCANIC SUBSTRATE1,2

Spatial studies of colonization and succession of soil algae and chemical analyses of the various soils on the cinde cone of Kilauea Iki in Hawaii Volcanoes National Park, Hawaii are outlined. There is a positive correlation between the diversity and quantity of soil algae with nutrient levels and organic matter accumulation in each locale. Three distinct edaphic-biotic zones existing in this area are differentially revealed by the soil chemical composition, quantity and diversity of soil algae, and as evident variations in higher plant growth and colonization. Varying colonization and successional phases of higher plant growth around standing and fallen tree snags killed by volcanic activity also reflect variations in the soil algal flora. These variations appear largely as a function of differential water interception, absorption, and retention as well as differential accumulation of organic matter, and the initiation of various biogeochemical cycles.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

转化淋巴细胞胰岛素受体酪氨酸蛋白激酶活性及细胞内源性底物的研究

本文对增殖期的淋巴细胞胰岛素依赖性酪氨酸蛋白激酶活性及内源性废物进行了分析研究。在纯化的健康人淋巴细胞中加入适量的植物血凝素(PHA),经过72h培养即成为转化淋巴细胞(增殖期淋巴细胞)。应用~(32)P参入实验,证实转化淋巴细胞胰岛素受体具有胰岛素依赖性的酪氨酸蛋白激酶活性,与未转化的对照组相比其活性增加约9倍。Scatchard分析表明转化后淋巴细胞膜表面胰岛素受体数增加3.5倍。应用抗酪氨酸磷酸酯抗体,对胰岛素作用前后的转化与未转化淋巴细胞内,酪氨酸残基磷酸化的蛋白进行了鉴定,结果表明:除了95kD受体β亚基自身磷酸化外,45kD蛋白质也明显磷酸化,我们命名它为PP45。我们认为PP45可能是淋巴细胞中胰岛素受体酪氨酸蛋白激酶的主要内源性废物,它的磷酸化是胰岛素信息传递过程级联反应的初始步骤。