Interaction of peptide boronic acids with elastase: circular dichroism studies

Boronic acid derivatives of good peptide substrates of the serine proteases cause slow-binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol Chem. 259:15106-15114, 1984). These inhibitors are thought to act as reaction-intermediate analogs. Three peptide boronic acids--Ac-Pro-boro-Val-OH, DNS-Ala-Pro-boro-Val-OH, and Ac-Ala-Ala-Pro-boro-Val-OH--were chosen for far-ultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitor (Ki = 0.27 microM) and the latter are slow-binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel beta-sheet by the peptide inhibitor itself indicate that there is no significant change in the secondary structure of the enzyme in either the initial or final inhibitor complex. A kinetic experiment confirmed that the slow-binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the slow binding.     

The Differentiation of Infection Structures as a Result of Recognition Events between some Biotrophic Parasites and their Hosts

Most uredospores of rust fungi develop infection structures in a typical pattern so that they can infect the host plant. The function of these infection structures is divided into the following three phases:

• 1 In the recognition phase, the germ tube recognizes the cuticle and the stoma. This process may occur independently from the host plant since copies of the cuticle induce similar reactions of the fungus. During fungal growth on the epidermis, unspecific stress responses of the plant are triggered.
• 2 In the signal phase, the fungal substomatal vesicle and infection hypha(e) contact the host cells within the leaf parenchyma. A signal from the host induces further development of the fungus. Haustorium mother cell differentiation is effected and haustorium formation is initiated. At the same time, the fungus suppresses the synthesis of stress metabolites by the plant.
• 3 In the parasitic phase, the fungus penetrates the host cell and complex interactions between host and parasite begin. A highly specialized interface around the haustorium develops presumably in order to allow a more efficient nutrient transfer from host to parasite. Eventual defence reactions of the plant, generally on the race-cultivar level, fail to be evoked or are suppressed in compatible combinations.

     

Changes in the nuclear structure in the radiation-sensitive CHO mutant cell, xrs-5

The structural organization of the cell nucleus was investigated by transmission electron microscopy in the radiosensitive Chinese hamster ovary (CHO) cell mutant, xrs-5 (D0 = 45 cGy), relative to parental K1 cells (D0 = 200 cGy). In 99% of all xrs-5 cells, the outer layer of the nuclear envelope was separated from the inner layer, while 96% of K1 cells had closely apposed layers. This separation of the inner and outer layers of the nuclear envelope in xrs-5 cells was not explained by an increased susceptibility of xrs-5 cells to osmotically induced changes because (1) xrs-5 cells retained the altered nuclear periphery even when several different fixation protocols were used and (2) xrs-5 cells were not more susceptible to cell lysis as measured by trypan blue dye exclusion or by the extracellular presence of lactate dehydrogenase. The difference in the morphological organization in the nuclear periphery of xrs-5 cells correlated with the radiation sensitivity of the cells; xrs-5 cells which spontaneously reverted to a radiation sensitivity similar to that of K1 cells also reverted to a nuclear morphology similar to that of K1 cells. The inner and outer layers of the nuclear envelope were retained in nuclear scaffolds isolated from K1 and xrs-5 cells, indicating that components of the nuclear periphery are part of the nuclear scaffold. These data show that xrs-5 cells have an altered nuclear periphery which correlates with the radiation sensitivity of the cells. The separation of the layers of the nuclear envelope may represent an altered template for repair of DNA damage at the nuclear scaffold and thus may play a role in the defective repair of X-ray-induced DNA double-strand breaks in xrs-5 cells.     

Effects of cryoprotectants on enzyme structure

The interaction between organic cosolvents and proteins is considered, especially from the point of view of effects on protein stability. It is concluded that each protein-cosolvent system constitutes a unique situation, making generalized predictions of expected effects difficult. Two classes of cosolvents are distinguished, based on the nature of their interactions with the protein surface. The thermodynamic instability to the system introduced by the presence of the cosolvent can be accommodated (i) by preferential exclusion of the cosolvent from the vicinity of the protein, (ii) by major structural changes of the protein, or (iii) by aggregation. Polyols tend to undergo preferential exclusion due to unfavorable interactions with nonpolar surface groups, whereas monohydric alcohols and other more hydrophobic cosolvents may undergo preferential exclusion due to adverse interactions with charged groups on the protein surface. Typical cosolvent effects on the structural and catalytic properties of enzymes are illustrated with data for ribonuclease and beta-lactamase with alcohol cosolvents. The relative hydrophobicity of the cosolvent is the major determinant of the effect of a cryosolvent on the enzyme stability and properties. Thus the position of the unfolding transition in cryosolvent will be decreased more by a more nonpolar cosolvent. Different cosolvents can have significantly different effects on the catalytic and structural properties of the same enzyme. Conversely the same cosolvent can have significantly different effects on similar proteins. The number and distribution of the nonpolar and charged groups on the protein's surface probably are the major determinants of the protein contribution to the solvent-protein interaction. The large temperature dependence of the rates of protein unfolding and refolding can be beneficially utilized in cryoprotectant studies of living cells.     

cDNA sequence of human beta-preprotachykinin, the common precursor to substance P and neurokinin A

The nucleotide sequence of cDNA encoding the human substance P precursor, beta-preprotachykinin (beta-PPT), has been determined. The source of mRNA was a human laryngeal carcinoid tumour that contained a high concentration of immunoreactive substance P. The human beta-PPT polypeptide is 129 amino acids long and contains regions encoding substance P and neurokinin A, each flanked by basic amino acid residues. Residues 72-107 of the human beta-PPT polypeptide encode the sequence of neuropeptide K, an N-terminally extended form of neurokinin A recently isolated from porcine brain.     

Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous and heterozygous fibroblasts

The esterification of cholesterol derived from human low density lipoprotein (LDL) or fetal bovine serum (FBS) was deficient in cultured fibroblasts from subjects with heterozygous and homozygous type C Niemann-Pick (NPC) disease. Failure to significantly esterify LDL-derived cholesterol resulted in abnormal accumulation of predominantly unesterified cholesterol in homozygous NPC fibroblasts. Compared with normal and homozygous fibroblasts, heterozygous NPC fibroblasts synthesized intermediate levels of cholesteryl ester during the initial 6 h of incubation with LDL. The rate of cholesterol esterification in heterozygous cells was normal when measured over a 24-h period of incubation with LDL. In addition to demonstrating a defect in cholesterol esterification, homozygous NPC fibroblasts accumulated more total cholesterol when incubated with LDL or FBS than normal fibroblasts accumulated. When heterozygous NPC fibroblasts were incubated with LDL or FBS, cellular accumulation of cholesterol reached levels that were high-normal or intermediary between levels observed in normal and homozygous NPC fibroblasts. The partial expression of these metabolic errors in the heterozygous genotype relevantly links these errors to the primary mutation of this disorder.     

The histidine permease gene (HIP1) of Saccharomyces cerevisiae

The histidine-specific permease gene (HIP1) of Saccharomyces cerevisiae has been mapped, cloned, and sequenced. The HIP1 gene maps to the right arm of chromosome VII, approx. 11 cM distal to the ADE3 gene. The gene was isolated as an 8.6-kb BamHI-Sau3A fragment by complementation of the histidine-specific permease deficiency in recipient yeast cells. We sequenced a 2.4-kb subfragment of this BamHI-Sau3A fragment containing the HIP1 gene and identified a 1596-bp open reading frame (ORF). We confirmed the assignment of the 1596-bp ORF as the HIP1 coding sequence by sequencing a hip1 nonsense mutation. Analysis of the amino acid (aa) sequence of the HIP1 gene reveals several hydrophobic stretches, but shows no obvious N-terminal signal peptide. We have constructed a deletion of the HIP1 gene in vitro and replaced the wild-type copy of the gene with this deletion. The hip1 deletion mutant can grow when it is supplemented with 30 mM histidine, 50 times the amount required for the growth of HIP1 cells. Revertants of this deletion mutant able to grow on a normal level of histidine arise by mutation in unlinked genes. Both these observations suggest that there are additional, low-affinity pathways for histidine uptake.