Characterization and isolation of a trypsin-like serine protease from a long-term culture cytolytic T cell line and its expression by functionally distinct T cells

We describe the characterization and purification of a trypsin-like serine protease isolated from cloned long-term culture cytolytic T cell line (CTLL AK). High amounts of proteolytic activity were isolated from extracts of CTLL AK after either nitrogen cavitation or detergent lysis. Trypsin-like protease was detected by using either the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester or a panel of low molecular amide substrates. The latter compounds were preferentially cleaved at the carboxyl termini of lysine and arginine residues. The enzyme activity was completely inhibited by two serine esterase inhibitors, diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and by aprotinin and meta-aminobenzamidine, which are known to block trypsin-like proteases. The pH optimum for CTLL AK-derived protease activity is 8 to 9. Analysis of the enzyme by gel filtration revealed that the cell-bound proteolytic activity was associated with a complex that could not be dissociated by treatment with Triton X-100. The CTLL AK-derived protease activity was found to reside in two proteins with relative molecular masses (Mr) of 32,000 and 40,000 daltons as determined by affinity labeling with [3H]diisopropylfluorophosphate and sodium dodecyl sulfate gel electrophoresis. High levels of enzyme activity were found in a panel of H-Y-specific cloned T cell lines with either cytolytic/suppressor (CTLL) or helper potential (THL), indicating a lack of correlation between trypsin-like protease activity and a particular T cell function. High enzyme activity was also detected in tumorigenic variants of CTLL. Furthermore, it was excluded that the trypsin-like activity detected was attributable to plasminogen activator activity. In contrast to cloned T effector cells and their in vitro or in vivo derived variants, considerably less activity was found in normal nonactivated or activated lymphocyte populations. The possible role of the trypsin-like serine protease in the function of T effector cells is discussed.     

Monoclonal antibodies to interferon-gamma inhibit interleukin 2-dependent induction of growth and maturation in lectin/antigen-reactive cytolytic T lymphocyte precursors

In this study we tested the effect of monoclonal antibodies (moAb) AN-18 to murine IFN-gamma on the generation of cytolytic T cells (CTL) from a homogeneous population of precursor cells (CTL-P). As responder cells, highly purified Lyt-2+ C57BL/6 lymph node T cells were used that had been positively selected by flow cytofluorometry on a cell sorter. Lyt-2+ cells were set up in bulk culture or in limiting dilution (LD) either with Con A or with P815 tumor cells as antigen and recombinant human interleukin 2 (rec.hIL 2) in the presence or absence of moAb AN-18 and tested for growth and development of CTL. The results show that moAb AN-18 but not the unrelated moAb AN-37 diminished or abrogated proliferative and cytolytic responses of Lyt-2+ lymphocytes to lectin and rec.hIL 2 in a dose-dependent manner. The inhibitory activity of the antibodies could be abolished by neutralizing moAb AN-18 with recombinant murine IFN-gamma (rec.mIFN-gamma) before their addition to culture. Kinetic analysis shows that the inhibitory effect of moAb AN-18 is only optimal when added at the beginning of culture or up to 48 hr after initiation. The frequencies of CTL-P responding either to Con A or to P815 tumor cells and rec.hIL 2 were reduced up to 10-fold in the presence of moAb AN-18. The inhibitory capacity of moAb AN-18 was also operative in cultures containing on the average one antigen-specific CTL-P. Together with the finding that activated CTL-P secrete IFN-gamma in response to rec.hIL 2 in a dose-dependent manner, the data suggest that endogenous IFN-gamma collaborates with exogenous IL 2 in the induction of CTL-P. The generation of CTL may therefore represent a case of autocrine growth regulation of normal lymphocytes, in which the same cell synthesizes and responds to its own factor.     

Seasonal variations in the pituitary response to LHRH in the brown hare (Lepus europaeus)

In the brown hare, fertile mating takes place from the beginning of December to September. Pituitary and ovarian response to a monthly i.v. injection of 5 micrograms LHRH was studied from September 1983 to October 1984 in 2 groups of 6 hares. The basal concentrations of LH remained undetectable until the end of January, rose from 0.23 +/- 0.14 ng/ml from February to a maximum of 1.44 +/- 0.57 ng/ml in July. LHRH injection was always followed by a release of LH. Between September and December, the LH value peaked 15 min after injection and returned to basal concentrations 2 h later. From January, this pattern altered and a second peak of LH appeared 2 h after injection. Peak levels 15 min after LHRH were around 10 ng/ml between September and December, increased from 47.0 +/- 8.0 ng/ml in January to 106 +/- 33 ng/ml in July and decreased in August (69.4 +/- 10.6 ng/ml). The values of the second peak rose from 11.0 +/- 2.2 ng/ml in January to 90.6 +/- 12.4 ng/ml between March and July and decreased in August (24.5 +/- 5.1 ng/ml). The LH surge induced by LHRH was always followed by a transient rise in progesterone. During the breeding season, this progesterone secretion increased considerably. Ovulation was possible between January and August and the number of ovulating females was maximum between March and July. The amount and duration of progesterone secretion during the resulting pseudopregnancies increased during the breeding season.     

The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study.

Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion.     

Bone mineral content of limb bones of male weanling rats subjected to 30 and 60 days of simulated increases in body weight

The purpose of this study was to subject groups of male rats each to a specific 10% simulated increase in body weight, ranging from 1.1 to 2.0 g. Constant centrifugation was employed. After 30 and 60 days, rats were sacrificed and perfused with 10% BNF. The humerus, radius, ulna, femur, and tibia were removed, cleared of all soft tissues, weighed, decalcified with EDTA, and reweighed. Bone mineral content (BMC) was determined using the formula: [(Wu - Wd) divided by Wu] X 100. Tukey's multiple range test was used. The data suggest that male weanling rats subjected to simulated increases in body weight, within the range used in this study, undergo enhanced BMC, a bimodal curve describing the relationship between BMC and simulated increases in body weight.     

Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.     

Slow charge movement in mammalian skeletal muscle

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)     

Requirements for the translocation of diphtheria toxin fragment A across lipid membranes

The translocation of the enzymatic moiety of diphtheria toxin, fragment A, across the membranes of pure lipid vesicles was demonstrated. A new assay, which employed vesicles made to contain radiolabeled NAD and elongation factor-2, was used to measure the appearance of the enzymatic activity of the A fragment in the vesicles. When the vesicles were exposed to a low-pH medium in the presence of diphtheria toxin, small molecules, such as NAD, escaped into the extravesicular medium, whereas large molecules mostly remained inside the vesicles. The vesicle-entrapped elongation factor-2 became ADP-ribosylated, indicating the entry of fragment A into the vesicle. The translocation of the A fragment depended upon the pH of the medium, being negligible at pH greater than 7.0 and maximal at pH 4.5. The entire toxin molecule was needed for function; neither the A fragment nor the B fragment alone was able to translocate itself across and react with the sequestered substrates. After exposure of the toxin to low pH, the entry of the A fragment was rapid, being virtually complete within 2-3 min at pH 5.5, and within 1 min at pH 4.7. Translocation occurred in the absence of any protein in the vesicle membrane. These results are consistent with the notion that the diphtheria toxin molecule enters the cytoplasm of a cell by escaping from an acidic compartment such as an endocytic vesicle.     

Electrostatic effect of trypsin binding on the hydrogen exchange rate of bovine pancreatic trypsin inhibitor beta-sheet NH's

The changes of H-D exchange rates upon protein-protein interactions are generally interpreted as a result of the changes of the dynamic properties of the proteins. The effect of trypsin binding on the H-D exchange kinetics of some trypsin inhibitor amide H's was reported (Simon et al., 1984). In this paper the electrostatic potential originating from the trypsin molecule is calculated at the positions of the studied amide H's in the trypsin-trypsin inhibitor complex. We conclude that the observed decrease of the exchange rates is mainly due to the electrostatic field of the trypsin molecule.     

Antigenic variation is associated with DNA rearrangements in a relapsing fever Borrelia

Borrelia hermsii, an agent of relapsing fever, undergoes antigenic variation in its host. Surface-exposed proteins with differing primary structures determine the serotype of each organism. Using amino acid sequence data from two of these variable proteins, we synthesized two mixed-sequence oligonucleotides and then used the oligonucleotides to probe mRNA and DNA of three isogenic serotypes of B. hermsii. In Northern blots the probes were specific for the mRNA of the homologous serotype. Southern blots revealed two classes of hybridizing fragments: those common to the three serotypes and those specific for a particular serotype. A serotype-specific DNA fragment, which had hybridized to both oligonucleotide probes, was cloned. Subsequent use of the cloned fragment as a probe provided further evidence that antigenic variation in B. hermsii is associated with DNA rearrangements and with occurrence of expression-linked copies of all, or part, of an antigen-specifying gene.