The cystatins: protein inhibitors of cysteine proteinases.

The last decade has witnessed enormous progress of protein inhibitors of cysteine proteinases concerning their structures, functions and evolutionary relationships. Although they differ in their molecular properties and biological distribution, they are structurally related proteins. All three inhibitory families, the stefins, the cystatins and the kininogens, are members of the same superfamily. Recently determined crystal structures of chicken cystatin and human stefin B established a new mechanism of interaction between cysteine proteinases and their inhibitors which is fundamentally different from the standard mechanism for serine proteinases and their inhibitors.     

Impact of protein-protein contacts on the conformation of thrombin-bound hirudin studied by comparison with the nuclear magnetic resonance solution structure of hirudin(1-51).

The impact of protein-protein interactions on the conformation of the N-terminal hirudin domain consisting of residues 1 to 51 in the X-ray crystal structure of a hirudin-thrombin complex was investigated through comparisons with the nuclear magnetic resonance solution structure of hirudin(1-51). The close overall similarity observed between these two structures contrasts with the behavior of the C-terminal 17-residue polypeptide segment of hirudin, which is flexibly disordered in solution but exhibits a defined conformation in the complex with thrombin. Localized structural differences in the N-terminal domain include that residues 1 to 3 of hirudin in the crystalline complex form a hydrogen-bonding network with thrombin that is reminiscent of a parallel beta-sheet. Moreover, the backbone conformation of residues 17 to 20 in the complex does not contain the characteristic hydrogen bond observed for the type II' reverse turn in the solution structure, and the side-chains of Ser19 and Val21 have significantly different orientations in the two structures. Most of these structural changes can be related directly to thrombin-hirudin contacts, which may also be an important factor in the mechanism of hirudin action. In this context, it is of special interest that other residues that also make numerous contacts with thrombin, e.g. Thr4, Asp5 and Asn20, have identical conformations in free hirudin and in the complex.     

Short term effects of oxidized ascorbic acid on bovine corneal endothelium and human placenta.

Studies on the toxic effects of dehydro-L-ascorbic acid (DHAA) have been extended to include evaluations over time periods up to 3 hr. and to test for specific effects on a membrane transport protein, a membrane-bound enzyme and a soluble intracellular enzyme. In studies on cultured corneal endothelial cells, DHAA concentrations of 1, 2, and 5 mM over 3 hr. had an inhibitory effect on subsequent uptake of DHAA present at a tracer level. Surviving fragments of human placenta and alkaline phosphatase activity of the placental brush-border membrane were susceptible to the effect of DHAA at a high concentration (10 mM). Because intracellular metabolism of DHAA was not affected, and an increase in membrane permeability was not detected, it is concluded that a specific membrane transport protein might be the site of DHAA-induced damage. These studies support the concept that the oxidized form of ascorbic acid (vitamin C) has potential toxic effects on biological systems and suggests that proteins that mediate transport and metabolism may be sites where DHAA causes damage.     

Enterohemolysin production is associated with a temperate bacteriophage in Escherichia coli serogroup O26 strains.

A temperate bacteriophage that determines the expression of enterohemolysin was isolated from Escherichia coli O26 strain C3888. The genetic determinant associated with enterohemolysin production (E-Hly determinant) was cloned from EcoRI-digested bacteriophage DNA in vector plasmid pUC8. pUC8 recombinant plasmid pEO19 carries a 3.7-kb EcoRI insert of phage DNA, and enterohemolysin was expressed in E. coli K-12 after transformation. Hemolysin-negative derivatives of pEO19 were generated by transposon mutagenesis with Tn1725. By subcloning, the phage E-Hly determinant was assigned to a 2,150-bp piece of DNA which is flanked by EcoRI and AccI restriction sites. The enterohemolysin-producing recombinant strains and wild-type strain C3888 express a 60-kDa protein which was detected in the bacterial outer membrane by Western immunoblotting. Biologically active enterohemolysin was detected only in bacteria grown to the stationary phase, and the hemolysin was not released into the culture medium. Lysis of erythrocytes was inhibited by 30 mM dextran 4, which functions as an osmotic protectant without destroying the enterohemolysin itself.     

The relationship between plasma free fatty acids and liver mitochondrial function in vivo

P/O ratio, state 3 and 4 respiration rates, and acceptor control index (ACI) were assessed in rat liver mitochondria following an overnight fast and single bout of treadmill exercise of 30-180 min. P/O was unaffected by fasting and 30 min of exercise; however, ACI was reduced because of an increase in state 4 respiration. Fasting, followed by running for 1 h or more decreased P/O approx. 40% and ACI by 50%, an effect that could be attributed to a reduction in state 3 respiration. The decrease in P/O was reversed 15 min after the cessation of exercise, whereas ACI remained depressed. All these functional alterations were mimicked by incubation of isolated mitochondria with palmitate and reversed by washing them with albumin. No direct correlation between plasma free fatty acids and the alterations in mitochondrial respiration was apparent. These data demonstrate that the decrease in the normal coupling of oxidation and phosphorylation in liver mitochondria produced by fasting/exercise is reversed rapidly in vivo. Furthermore, it is apparent that, if fatty acids act as a regulatory agent under these conditions, they do not do so solely on the basis of their plasma concentration.     

Crystallization,crystal structure analysis and molecular model of the third domain of Japanese quail ovomucoid,a Kazal type inhibitor

The third domain of Japanese quail ovomucoid, a Kazal type inhibitor, has been crystallized and its crystal structure determined at 2.5 Å resolution using multiple isomorphous replacement techniques. The asymmetric unit contains four molecules. In the crystal the molecules are arranged in two slightly different octamers with approximate D4 symmetry. The molecules are held together mainly by interactions of the N-terminal residues, which form a novel secondary structural element, a β-channel.The molecule is globular with approximate dimensions 35 Å × 27 Å × 19 Å. The secondary structural elements are a double-stranded anti-parallel β-sheet of residues Pro22 to Gly32 and an α-helix from Asn33 to Ser44. The reactive site Lys18-Asp19 is located in an exposed loop. It is close to Asn33 at the N terminus of the helical segment. The polypeptide chain folding of ovomucoid bears some resemblance to other inhibitors in the existence of an anti-parallel double strand following the reactive site loop.     

Effect of alcohol on microsomal cortisol 4en-5 alpha-reductase in the liver of rats fed on a standard or low protein diet

Alcohol feeding (40% of total calories over a period of 9 days) increases microsomal cortisol-5 alpha-reductase activity in rat liver distinctly. It is assumed that this is an adaptive response to an increased release of cortisol caused by alcohol administration. Cortisol-5 alpha-reductase activity is decreased to one third of control values in rats fed an isocaloric, low protein diet. The response to alcohol feeding is susta ined in these animals. Phenobarbital treatment (80 mg/kg x day) stimulates 5 alpha-reduction of cortisol per g of microsomes almost twofold. The activity calculated per total liver increases 4-fold. Alcohol administration has no additional effect on cortisol-5alpha-reductase in phenobarbital-treated rats.     

The use of subunit exchange chromatography for the group specific fractionation of histones

Starting from the histone mixture obtained from calf thymus, the arginine rich fraction ARE⁺⁾ was coupled to organomercurial agarose via a mercaptide bond to one of its cysteines. ARE-agarose proved to be useful for a large scale affinity chromatographic separation of whole histone. In 1M NaCl, pH 4.5, highly pure histone fractions could be eluted with an urea gradient revealing increasing affinity towards ARE in the order: KAP < KAS < LAK < (ARE)_n(GRK)_n < GRK < ARE.