A high molecular weight protease in the cytosol of rat liver. II. Properties of the purified enzyme

The properties of a soluble endoprotease from rat liver were studied. The enzyme was purified in a latent form. It sedimented as a single component with a sedimentation coefficient (S(0)20,w) of 19.8 S. Measurement by quasi-elastic light scattering gave a diffusion coefficient (D(0)20,w) of 2.5 X 10(-7) cm2 X s-1 and an effective hydrodynamic radius of 85 A. The enzyme had an unusually high molecular weight, estimated as 743,000 by sedimentation equilibrium and 722,000 by sedimentation velocity and diffusion measurements and as 760,000 by a recently developed low-angle laser light scattering method. Judging from electron microscopic observation and the calculated frictional and axial ratios, the enzyme molecule is disc-shaped. Analysis of the far-ultraviolet circular dichroic spectrum showed that the enzyme contains 50% alpha-helical, 25% beta-sheet, and 15% unordered structures with 10% beta-turns. The isoelectric point of the enzyme is 5.0. These properties indicate that the purified enzyme is a homogeneous molecule. In addition, the enzyme is a simple protein since it contains no measurable amounts of nucleic acid carbohydrate or lipid.     

A unique trypsin-like protease associated with plasma membranes of rat liver

One way in which serum promotes survival of primary cultured hepatocytes is by inhibiting plasma membrane protease (Nakamura, T., Asami, O., Tanaka, K., and Ichihara, A. (1984) Exp. Cell Res. 155, 81-91). One of these proteases was solubilized from the plasma membranes of rat liver with 4% octyl glucoside and purified to a homogeneous state by affinity chromatography on bovine pancreatic trypsin inhibitor linked to Sepharose 4B. The protease had an apparent Mr = 120,000 by Sephacryl S-200 gel filtration and the Mr of its subunits was 30,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It appeared to be a glycoprotein. A high concentration of detergent was necessary to keep the protein soluble. The purified enzyme readily hydrolyzed synthetic tripeptide nitroanilides at sites adjacent to Arg or Lys residues, but did not degrade synthetic substrates of chymotrypsin, elastase, or aminopeptidase. It showed endopeptidase activity, hydrolyzing various proteins such as casein, hemoglobin, and denatured albumin. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bovine pancreatic trypsin inhibitor, leupeptin, antipain, and alpha 1-antitrypsin, but not by chymostatin, elastatinal, or inhibitors of carboxyl, thiol, or metallo proteases, suggesting that it is a seryl trypsin-like protease. This protease was found in plasma membranes of rat and mouse liver and in small amounts in those of kidney, but not in those of brain, red cells, Ehrlich ascites tumor, or two Morris hepatomas, suggesting that it may be involved in differentiated functions of normal hepatocytes.     

Tissue distribution of rat angiotensinogen mRNA and structural analysis of its heterogeneity

The tissue distribution and the structural heterogeneity of the rat angiotensinogen mRNA have been investigated with the aid of a previously cloned cDNA as well as a genomic DNA for rat angiotensinogen as analytical probes. The angiotensinogen mRNA is expressed not only in the liver but also in various tissues including the brain, kidney, adrenal gland, ovary, and lung. The relative levels of the mRNA in the above tissues have been estimated to be 3-4, 20-30 (for the next three tissues), and around 100 times less than that in the liver, respectively. The mRNAs in both hepatic and extrahepatic tissues are encoded by a single gene in the rat genome. At least four different size classes of the angiotensinogen mRNA that start with a single 5' terminus and differ only in the lengths of their 3'-untranslated regions have been identified, and these multiple mRNA species are most likely generated by using the polyadenylation signals AAUAAA and AUUAAA found 10-30 nucleotides upstream from the four polyadenylation sites. Because the structures of these multiple mRNA species do not vary among the tissues of the liver, brain, and kidney, angiotensinogen synthesized locally is structurally identical to that produced in the liver and may have some biological roles independent of the circulating angiotensinogen, mainly derived from the liver. In addition, the sequence of the 5'-flanking region of the angiotensinogen gene has been determined, and some features common to other steroid hormone-responsive genes have been discussed.     

tert–butylhydroperoxide—Induced toxicity in isolated hepatocytes: Contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also, occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide–induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

Effect of substance P on neurally mediated contraction of rabbit airway smooth muscle

The neuromodulatory action of substance P (SP) was investigated in isolated rabbit tracheal smooth muscle (TSM) segments contracted with electrical field stimulation (ES). The tissues were placed in organ baths containing modified Krebs-Ringer solution and stimulated at a constant voltage (8 V; 24.5 mA) and pulse duration (2 ms) with ES frequencies ranging from 1 to 100 Hz. In the presence of SP, there occurred a dose-dependent augmentation of the TSM contractile response to any given ES, with the maximal effect of SP obtained at a dose of 10(-7) M. Accordingly, with the administration of 10(-7) M SP, the ES frequency-response relationship was altered so that 1) the mean (+/- SE) maximal tension (Tmax) induced by ES significantly increased (P less than 0.02) from a base-line value of 273 +/- 53 to 402 +/- 45 g/g TSM; and 2) the mean (+/- SE) log ES frequency producing 50% of Tmax (ES50) significantly decreased from a base-line value of 1.278 +/- 0.069 to 1.102 +/- 0.070 Hz (P less than 0.01). In contrast to these effects on ES-induced contraction, SP administration did not affect the TSM contractile response to administered methacholine chloride (10(-8) to 10(-3) M). On the other hand, the effects of SP on ES-induced contraction were independently blocked by the cholinergic antagonist, atropine (10(-6) M); the neurotoxin, tetrodotoxin (10(-6) g/ml); and the SP antagonist, D-Arg1,D-Pro2,D-Trp7,9,Leu11-SP (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Basic studies of cryochemotherapy in a murine tumor system

The combined effect of cryosurgery and anticancer drugs (cryochemotherapy) was studied in an experimental B16 melanoma/BDF1 tumor system. Vascular volume and vascular permeability after cryosurgery of normal skin and the tumor were measured by using 51Cr-labeled red blood cells and 125I-labeled serum albumin. The vascular volume and vascular permeability of both the normal vessels and the tumor vessels greatly increased immediately after cryosurgery, and their vascular volume decreased to less than the normal level within a few hours. However, the tumor vessels showed less dilatation and increase in permeability than the vessels of normal tissue. There was a difference in functional characteristics in response to cryoinjury between the normal vessels and the tumor vessels. The anticancer drugs, peplomycin and adriamycin, were administered intraperitoneally in combination with cryosurgery. When peplomycin was administered 5 min, 1 hr, and 3 hr after cryosurgery, the drug concentration in the frozen tumor was higher than that in the untreated tumor. But when administered 1 hr before cryosurgery, peplomycin was not trapped in the tumor. Trapping of adriamycin was not observed after the same treatment. In cryochemotherapy, it is necessary to administer the appropriate drug at the appropriate time. However, the trapping of the anticancer drug results in a high concentration and lasts for a long time, so that cryochemotherapy is expected to be a new mode of cancer therapy, particularly as a multidisciplinary treatment for cancer.     

Synthesis of a nonacosapeptide (beta-fragment) corresponding to the N-terminal sequence 1-29 of human liver metallothionein II and its heavy metal-binding properties

A nonacosapeptide (beta-fragment) corresponding to the N-terminal sequence 1-29 of human liver metallothionein II was synthesized by the fragment condensation method. The Cd-binding ability of the beta-fragment was much stronger than that of cysteine as thionein and synthetic alpha-fragment corresponding to the C-terminal sequence 30-61 of human liver metallothionein II. Both the alpha- and beta-fragments bound preferentially to Cu ions rather than Cd ions.     

Interleukin 2 acts directly on a Tac-positive cloned B cell line established from a patient with adult T cell leukemia

A Tac-positive B cell line termed K3B was established from a patient with adult T cell leukemia (ATL). This cell line had EBNA antigen and human T cell leukemic virus (HTLV) provirus besides B1 antigen and surface immunoglobulin. A cloned Tac-positive B cell line termed K3B01 was obtained from K3B by the limiting dilution method. The K3B01 cells were shown to absorb IL 2 activity in a tonsillar IL 2 preparation. By using this cloned cell line and a purified recombinant IL 2 preparation, it was shown that the proliferation of K3B01 cells was enhanced by the addition of recombinant IL 2. Moreover, this response was inhibited by anti-Tac antibody. These results demonstrate definitively that IL 2 acts directly on B cells through IL 2 receptors on them.     

X-ray crystallographic and chromatographic characterization of the crystals of Ca2+-calmodulin complexed with bee venom melittin

Crystals of calmodulin complexed with both Ca2+ and melittin, a peptide from bee venom, have been grown from 2-methyl-2,4-pentanediol solution by using the hanging drop method of vapour diffusion. The crystals belong to space group P2(1)2(1)2(1) with a = 97.3(9) A, b = 56.5(0) A, c = 33.4(9) A and Z = 4. Analyses of the dissolved crystals by high performance liquid chromatography show that the crystals contain a 1:1 complex of calmodulin and melittin. An asymmetric unit contains one such complex and the solvent content of the crystals is 47.5% (v/v).