Picolyl esters of amino acids are suitable inverse substrates for trypsin. These esters can be used for peptide synthesis catalyzed by trypsin. Study of hydrolysis of non-specific substrates for trypsin permits to conclude, that hydrogen band in P'2 subsite is very important for their reactivity. Carboxamido methyl esters (CAM-esters) are the most suitable substrates for trypsin catalyzed peptide synthesis. Using CAM-esters and trypsin several di-,tri- and tetra-peptides were synthesized with good yields.
The inactivation of soybean lipoxygenase-1 and of rabbit reticulocyte lipoxygenase by five selected acetylenic fatty acids was studied. In all cases the inactivation was time-consuming and depended on the concentration of the inactivator. The inactivation kinetics was measured and the data were fitted to a kinetic model based on the assumption of catalytic self-inactivation. The kinetic constants (Km-value and inactivation rate k2) calculated indicated that 7,10,13-eicosatrienoic acid was the most powerful inactivator for the soybean enzyme followed by 8,11,14-eicosatrienoic acid. The occurrence of an additional triple bond between C-4 and C-5 or between C-5 and C-6 strongly reduced the suicidal rate. With the reticulocyte enzyme, only small differences in the reactivities towards various acetylenic fatty acids have been observed.
ts85, a cell-line that harbors a mutant thermolabile ubiquitin-activating enzyme, E1, fails to degrade short-lived proteins at the restrictive temperature (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). It is not known whether the ubiquitin system is also involved in the degradation of long-lived proteins. In the present study we show that upon shifting the mutant cells to the restrictive temperature, there is no change in the rate of degradation of long-lived proteins. In contrast, shifting the wild-type cells (FM3A) to the high temperature is accompanied by a 2-fold increase in the rate of proteolysis of this group of proteins. This heat-induced accelerated degradation can be completely inhibited by NH4Cl and chloroquine. Similarly, exposure of the cells to starvation, a stimulus that activates the autophagic-lysosomal pathway, has no effect on the degradation of long-lived proteins in the mutant cells following inactivation of E1. Under the same conditions, the degradation rate in the wild-type cells increases almost 4-fold. A revertant of the ts85 cells behaved in a similar manner to the wild-type cells. Analogous results were obtained using a different cell line that also harbors a thermolabile E1 (ts20) (Kulka, R. G. et al. (1988) J. Biol. Chem. 263, 15726-15731). Cycloheximide and 3-methyladenine, inhibitors of formation of autophagic vacuoles, suppress the heat-induced accelerated degradation in the wild-type cells. Taken together, the results suggest that: 1. heat stress induces enhanced degradation of intracellular proteins, 2. the process occurs most probably in autophagic vacuoles, 3. activation of ubiquitin is required for enhanced degradation to occur, and 4. the activation is involved most probably in formation of the autophagic vacuoles.
The influence of D-glucose on electrolyte and water transport in the distal ileum and proximal colon of the rabbit in vitro and in vivo has been studied. The magnitude of electrolyte and water transport was found to differ in vitro and in vivo. The replacement of mannitol by D-glucose in the intestinal lumen had no effect on ileal and colon transport of the ions studied, i.e., movement of the ions was independent of the supply of exogenous glucose. The distal ileum and the proximal colon of the rabbit in vivo showed a net secretion of bicarbonate, aimed at neutralizing the cecal contents.
The gene structures of rat cathepsins H and L have been determined. Cathepsin H gene spans more than 21.5 Kbp and comprises more than 12 exons. On the other hand, cathepsin L gene spans 8.5 Kbp and comprises 8 exons. In both genes, two intron insertion positions are conserved at the amino acid level. Both enzymes, therefore, diverged from a common ancestral gene. In the 5'-upstream region of the cathepsin L gene, no TATA box, one CAAT box and some SP-1 binding sites exist, common with other lysosomal enzyme genes. Furthermore, two AP-2 binding sites exist: a promoter under the control by the tumor promoter (TPA) and cAMP, and a cAMP response element (CRE). These results suggest that the cathepsin L gene expression is controlled by these factors.
Our recent researches toward enzymic condensation of long chain peptides are discussed. These involve two methods of peptide C-terminal activation and transpeptidation catalyzed by clostripain, trypsin and their derivatives.
In medical research and clinical practice, the increasing application of filtration measurements, to characterize the erythrocyte deformability has aroused an intensive discussion about possible erroneous interpretations of measured values (filterability indices). In the present paper the influence of white blood cells (WBC) on these indices is described and discussed in relation to a mathematical model of the filtration process. We found for two gravity filtration systems, using cellulose filters and concentrated erythrocytes suspensions sensitivities of their filterability indices of (1 +/- 0.1)% per 100 WBC/microliter (initial filtration rate measurement) and (3 +/- 0.3)% per 100 WBC/microliter (hemofiltrometer). In comparison to Nuclepore filtration systems with sensitivities of 5-12% per 100 WBC/microliters, cellulose filters were much less sensitive to WBC. The necessity, advantages and disadvantages of additional procedures for WBC removal are discussed. A standardization of residual WBC-count in limits of 500 +/- 300 WBC/microliters at a hematocrit of 60% is proposed. Fitting of the filtration curves of the hemofiltrometer to a model function may either improve the accuracy of estimation of filterability indices or divide them into two model parameters with different WBC-sensitivities: the initial filtration rate parameter having a low WBC-sensitivity ((0.9 +/- 0.2)% per 100 WBC/microliter) and the occlusion related parameter having a 12 times higher sensitivity ((11 +/- 1)% per 100 WBC/microliter). Interpretation, advantages and disadvantages of the model parameters are discussed also in comparison to the filterability index.
The venoms of most Crotalidae snakes contain metalloproteinases which are the agents responsible for the production of venom-induced hemorrhage via proteolytic destruction of capillary basement membranes. Prevention of hemorrhage by administration of antiserum is generally not totally effective against damage at the site of envenomation. Therefore, we have investigated alternate methods for the alleviation of hemorrhage by inhibition of the proteolytic activity of the hemorrhagic toxins. The first approach involves the synthesis of carboxyalkyl peptide inhibitors in which the peptide moiety is modeled on the substrate specificity of the toxins. With this approach we have determined that the carboxypentyl group for interaction with the active site Zn++ ion is most effective. Also, longer peptide moieties enhance the inhibitors' activity giving K(i)'s in a range of 10(-6) M. Our second approach to hemorrhagic toxin inhibition was to search for the presence of endogenous inhibitors against the toxins in the venom. From the crude venom we have isolated several pyro-glutamate containing peptides, two of which are relatively good inhibitors of the toxins. The isolation and characterization of the endogenous toxin inhibitors as well as the synthetic inhibitors may ultimately serve as a basis for new, effective treatments against venom-induced hemorrhage.
The interaction of delta (Ser50)-hirudin with alpha-thrombin has been investigated. Deletion of Ser50 of r-hirudin caused a 2.7 fold increase of the Ki for its complex with alpha-thrombin. Determination of the rate constants kon and koff for complex formation showed that this effect was mainly due to a change in koff.
Human polymorphonuclear leukocytes mainly protect the body against invading microorganisms. Therefore, cells must migrate to the site of infection, whereby they traverse the endothelial cell layer of the blood vessels, basement membranes and the connective tissue. We investigated the unknown mechanism of penetration of basement membranes by scanning electron microscopy. Our findings indicate a three-step mechanism--adhesion, locally limited proteolytic degradation of matrix components and mechanical loosening of the matrix. Finally, cells locomote in the stroma tissue to the site of infection. These processes are induced by formyl-peptides amongst others. In addition, an increased release of collagenase induced by formyl-peptides was observed. In contrast, incubation with prostaglandin F2 alpha led to significantly higher levels of secreted collagenase, while elastase failed to be detected in the supernatant of stimulated cells. Since it has been shown that collagenase is secreted in a latent form, we have focussed our attention on the activation mechanism of the proenzyme, which was investigated by determination of the N-terminal sequences of intermediate and final, activated forms.
Various polymeric oxidation products of polyphenols strongly inhibited the purified lipoxygenase of rabbit reticulocytes, whereas the prostaglandin H synthase of sheep vesicular gland was only weakly inhibited. The oxidized polymeric preparations of caffeic acid, 2,5-dihydroxytoluene and 3,4-dihydroxytoluene were the most potent lipoxygenase inhibitors. Since such preparations are also known to inhibit herpes simplex virus in an early stage of the virus-cell interaction it is proposed that lipoxygenase metabolites are involved in virus-induced changes in the cell metabolism.
Various inactivation methods were applied to lipoxygenases from soybean (isoenzyme 1) and rabbit reticulocytes to compare the inactivation behaviour of both enzymes and to elucidate the state of the iron which is known to be involved in the catalytic reaction of lipoxygenases:1. Titration of the enzyme with mercury compounds shows that there are one or two SH groups responsible for the loss of activity in the presence of mercury. The SH groups seem not to be involved in the tight iron binding.2. Inactivation by chelating agents such as o-phenanthroline or batho-phenanthroline sulfonic acid occurs only in the presence of reducing agents (mercaptoethanol and ascorbic acid). Our data support a co-oxidation mechanism. The complexation of iron by chelators is not the rate-limiting step. Both lipoxygenases show a very similar behaviour in this respect despite the fact that the reticulocyte enzyme requires the addition of trace amounts of copper ions for efficient inactivation.3. Release of iron from the enzyme is also achieved by denaturation with guanidinium hydrochloride (Gu-HCl). In all cases, inactivation and release of iron were irreversible processes.4. A sequence comparison for both animal and plant lipoxygenases shows strongly conserved amino acids, especially histidines and hydrophobic residues, which possibly may be involved in iron complexation and substrate binding.
Lipoxygenase was purified from wheat kernels by means of ammonium sulfate precipitation, gel chromatography on Sephadex G-200 and anion exchange chromatography on DEAE-Sephadex A-50. Arachidonic acid was mainly converted by the wheat lipoxygenase to 5D-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5D8-HPETE) with other HPETE isomers including 8-HPETE being minor products. At higher concentrations of lipoxygenase, multiple oxygenation products such as 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and, to a lower extent, 8,15-diHETE and lipoxin isomers were detected after reduction of the hydroperoxy derivatives primarily formed. Similar results were obtained with 5D8- or 15L8-hydroxyeicosatetraenoic acid as substrate. Moreover, evidence was obtained for leukotriene A4 synthase activity of the wheat lipoxygenase.
A competitive ELISA for osteocalcin in 10 microliters samples has been developed with alkaline phosphatase as a marker enzyme. The assay for osteocalcin has an inter-assay and intra-assay variability of 13% and 12%, respectively; the limit of detection is 3.6 pg per assay. The assay was used to measure osteocalcin serum levels in patients with chronic hemodialysis and several bone diseases. The method for the purification of osteocalcin is also described.
Alterations in the erythrocyte rheology and the contents of activated free radical oxidation products (conjugated dienes, products of thiobarbituric acid and Schiff bases) in the acute phase of experimental thermic injury of the skin were studied. Erythrocyte flexibility reduction and erythrocyte aggregation increase correlated with elevated amounts of free radical oxidation products. Alpha-tocopherol avoided the accumulation of free radical oxidation products and improved both antioxidant defence and erythrocyte rheology. Thus we suppose that free radical oxidation products probably participate in the pathogenesis of erythrocyte rheology disturbances after thermic trauma.
Small amounts of serum added to lens epithelial cells in primary culture give rise to an initial retraction of the epithelial sheet and a following stimulation of spreading. It could be shown that albumin or a component tightly bound to albumin is responsible for triggering off these early events of reactivation in culture. The stimulation of spreading is accompanied by an increase of protein biosynthesis. The albumin-produced stimulation of both spreading and protein synthesis are counteracted by adenine or adenosine in the presence of inorganic phosphate. The albumin as well as the purine effects disappear after subculturing of the cells.
The steady state kinetics of 6-phosphofructo-1-kinase was determined in a cell-free extract obtained from a yeast mutant (DFY 250) and compared with the kinetic properties of the enzyme of a wild-type strain (DFY 1). 6-Phosphofructo-1-kinase from the DFY 250 strain shows a complex kinetic behaviour, which is qualitatively similar to, but quantitatively different from, that of normal yeast 6-phosphofructo-1-kinase. The mutant enzyme has a lower affinity to its activators fructose 6-phosphate, fructose 2,6-bisphosphate and AMP. The inhibiting effect of ATP on the mutant 6-phosphofructo-1-kinase is substantially weaker than on the wild-type enzyme. A complex interaction between fructose 6-phosphate and fructose 2,6-bisphosphate at the 6-phosphofructo-1-kinase from strain DFY 250 is reflected by a remarkable substrate inhibition by fructose 6-phosphate even at saturating fructose 2,6-bisphosphate. The kinetic data were fitted to different variants of the Monod-Wyman-Changeux model by nonlinear regression analysis. It turned out that the influence of fructose 6-phosphate, ATP, AMP and fructose 2,6-bisphosphate on the activity of 6-phosphofructo-1-kinase from wild-type and DFY 250 strain could be described by rate equations of essentially the same structure.
A simple and rapid method for the isolation of bovine heart mitochondrial adenosine 5'-triphosphatase (F1-ATPase) was developed. Mitochondria were purified by differential centrifugation and stored frozen. After thawing. F1-ATPase was released by treatment with chloroform. Purification of the enzyme was achieved by polyethylene glycol precipitation followed by chromatography on Procion Navy H-ER beaded cellulose in the presence of MgCl2. F1-ATPase was eluted by ATP in the absence of MgCl2. The purity of the enzyme was proved by SDS-polyacrylamide-gel electrophoresis. The purified F1-ATPase showed slightly non-hyperbolic kinetics towards ATP and nearly complete inhibition in the presence of millimolar concentrations of ADP.
