Further inhibition studies on guanidinobenzoatase, a trypsin-like enzyme associated with tumour cells

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasminogen activator, plasmin, thrombin and a newly described tumour associated enzyme specific for guanidino phenylalanine residues. These conclusions have been derived from inhibition studies employing 4-methyl-p-guanidinobenzoate as substrate. Three active site titrants for trypsin have been shown to be good substrates for guanidinobenzoatase. A new active site titrant for trypsin, rhodamine bisguanidinobenzoate, can also be used to assay guanidinobenzoatase in a stoichiometric manner. This active site titrant can be employed to label guanidinobenzoate on the surface of leukaemia cells.     

Inhibition studies of soybean trypsin-like enzyme

The results of inhibition studies of soybean trypsin-like enzyme (STLE) by substrate analogues (derivative of arginine) suggested that a net negative charge exists at or near the substrate binding region of the enzyme. On hydrolysis of substrates, this negative charge seems to repel the products from the substrate binding region and facilitate the turn-over of substrates. From the data on inhibition by various amidines, guanidines, and amines, some information about the structure of the hydrophobic binding pocket of STLE was obtained. The inactivation of STLE by irreversible inhibitors, diisopropylfluorophosphate (DFP) and tosyl-lysine chloromethyl ketone (Tos-Lys-CH2Cl), was decreased by competitive inhibitors. This means that these irreversible inhibitors bind with residues at the substrate binding region, probably serine and histidine residues, respectively.     

Further studies on a British form of pea early-browning virus

An isolate of pea early-browning virus from Britain (PEBV (B)) has tubular particles most of which are either about 103 or 212 mμ long with sedimentation coefficients of 210 and 286 S respectively. Both types show cross-banding at intervals of 2.5 mμ. Virus preparations containing only the shorter particles were not infective. PEBV (B) was transmitted to pea seedlings by both adult and juvenile Trichodorus primitivus (de Man) (Nematoda) and persisted for 32 days in T. primitivus kept without plants. In two experiments T. primitivus failed to transmit a Dutch isolate (PEBV (D)), which is distantly related serologically to PEBV (B). PEBV (B) was transmitted by nematodes to cucumber roots more readily in soil at 20d? than at 24d? C., and more readily at 24d? than at 29d? C. When transmitted by inoculation of sap, PEBV (B) and PEBV (D) caused similar symptoms in some pea varieties but differed in virulence towards others. Thirty-one varieties resistant to natural infection with PEBV in The Netherlands were susceptible to PEBV (B) when manually inoculated with sap or when grown in naturally infested soil from one site; twenty-six of these varieties did not become infected in soil from a second site, in which several other varieties that are susceptible in The Netherlands were infected. Varieties should therefore be tested for resistance by growing them on many infested fields. All but one of the pea varieties resistant to PEBV in The Netherlands became infected with the English form of tomato black ring virus when grown in soil containing infective Longidorus attenuatus Hooper.     

Morphometric studies on hamster testes in gonadally active and inactive states: light microscope findings

This study provides quantitative information on the testes of seasonally breeding golden hamsters during active and regressed states of gonadal activity. Seminiferous tubules occupied 92.5% of testis volume in adult gonadally active animals. Leydig cells constituted 1.4% of the testicular volume. The mean volume of an individual Leydig cell was 1092 microns 3, and each testis contained about 25.4 million Leydig cells. The volume of an average Sertoli cell nucleus during stage VII-VIII of the cycle was 502 microns 3. A gram of hamster testis during the active state of gonadal activity contained 44.5 million Sertoli cells, and the entire testis contained approximately 73.8 million Sertoli cells. Testes of the hamsters exposed to short photoperiods for 12-13 wk displayed a 90% reduction in testis volume that was associated with a decrease in the volume of seminiferous tubules (90.8% reduction), tubular lumena (98.8%), interstitium (72.7%), Leydig cell compartment (79.3%), individual Leydig cells (69.7%), Leydig cell nuclei (50.0%), blood vessels (85.5%), macrophages (68.9%), and Sertoli cell nuclei (34.1%). The diameter (61.1%) and the length (36.8%) of the seminiferous tubules were also decreased. Although the number of Leydig cells per testis was significantly lower (p less than 0.02) after short-photoperiod exposure, the number of Sertoli cells per testis remained unchanged. The individual Sertoli cell in gonadally active hamsters accommodated, on the average, 2.27 pre-leptotene spermatocytes, 2.46 pachytene spermatocytes, and 8.17 round spermatids; the corresponding numbers in the regressed testes were 0.96, 0.20, and 0.04, respectively. The striking differences in the testicular structure between the active and regressed states of gonadal activity follow photoperiod-induced changes in endocrine function and suggest that the golden hamster may be used as a model to study structure-function relationships in the testis.     

Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP binding to an inactive form of the enzyme

The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70-80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half-maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the trimer. Also, the dCTP complex of the inactive H121A was found to have residues 120-125 in this new conformation, indicating that it renders the enzyme inactive. The C-terminal fold was found to be disordered for both new complexes. We suggest that the cooperative kinetics are imposed by a dTTP-dependent lag of product formation observed in presteady-state kinetics. This lag may be derived from a slow equilibration between an inactive and an active conformation of dCTP deaminase represented by the dTTP complex and the dUTP/dCTP complex, respectively. The dCTP deaminase then resembles a simple concerted system subjected to effector binding, but without the use of an allosteric site.     

Inductions of superoxide dismutases in Escherichia coli under anaerobic conditions. Accumulation of an inactive form of the manganese enzyme

Escherichia coli growing anaerobically respond to NO3- with a 3-fold induction of the iron-containing superoxide dismutase. Mutants lacking nitrate reductase do not show this response. Anaerobically grown cells also contain an inactive form of the manganese-containing superoxide dismutase (MnSOD) which can be activated by addition of Mn(II) salts in the presence of acidic guanidinium chloride, followed by dialysis against neutral buffer. Direct addition of Mn(II) to a neutral solution of the inactive MnSOD does not impart activity. This inactive MnSOD thus behaves as would the apoenzyme or the enzyme bearing a metal other than Mn(II) at its active sites. Terminal electron acceptors, such as NO3- or trimethylamine N-oxide, increase the amount of inactive MnSOD produced by anaerobic E. coli. Paraquat, which is itself ineffective in this regard, markedly augments the effect of these terminal electron acceptors. It appears that flow of electrons to sinks such as NO3- or trimethylamine N-oxide, facilitated by paraquat, is sufficient to elicit biosynthesis of the MnSOD protein and that O2- is not needed for this process. Yet, oxygenation and concomitant O2- production do appear important for the insertion of manganese into the growing MnSOD polypeptide, possibly because O-2 oxidizes Mn(II) to Mn(III), and the latter is the valence state most effective in combining with the apoenzyme.     

Kinetic investigation of soybean trypsin-like enzyme catalysis

Various esters and amides of benzoylarginine and of benzyloxycarbonylarginine were subjected to enzymic hydrolysis at pH 8.5 and 7.2 by soybean trypsin-like enzyme (STLE). The kcat values for the hydrolysis of esters and amides were essentially identical regardless of the kind of leaving group. These results suggest that the STLE-catalyzed hydrolysis of ester and amide substrates proceeds via an acylenzyme intermediate and that the deacylation step is rate-determining. Hydrolysis of various 4-methylcoumaryl-7-amides of varying chain length and amino acid sequence was carried out at pH 8.5. Analysis of kinetic parameters revealed that STLE does not exhibit any remarkable subsite requirement, but somewhat preferentially hydrolyzes shorter substrates. These observations are consistent with the fact that STLE does not hydrolyze protein substrates or oxidized insulin B chain but hydrolyzes oligopeptides (Nishikata, M. (1984) J. Biochem. 95, 1169-1177). It is possible that the active site of STLE is located at a deep position in the enzyme molecule. From the pH dependency of kcat/Km, the participation of a histidine residue in the catalytic process of STLE was suggested.     

Cell density dependent regulation of phenylalanine hydroxylase activity in hepatoma cells : Evidence for both an active and inactive enzyme form

The cell density dependent regulation of phenylalanine hydroxylase activity in Reuber hepatoma (H4) cells growing in monolayer culture has been examined in detail. We found that 48 h or more after subculture phenylalanine hydroxylase activity in the cells is an exponential function of cell density (cells/cm²). No discontinuity in the relationship is seen with the formation of a confluent monolayer.A rapid loss or a rapid gain in enzyme activity in the cells is observed after diluting or concentrating the cell cultures. The two processes appear qualitatively different. The loss in activity is a first order process which starts at the time of subculture with the rate of loss dependent on the density of subculture. The gain in activity begins 6–8 h after subculture to a higher density; it can be blocked by cycloheximide and has a maximum rate of increase that is about 10% of the maximum rate of loss of activity.Using immunochemical procedures, we found the same amount of phenylalanine hydroxylase associated antigen in Reuber cells from low density as from high density cultures, over a range of phenylalanine hydroxylase specific activities from 0.2 to 4.2. After concentrating cells to a higher density, no increase in enzyme antigen was observed, despite a several-fold increase in enzyme activity and a requirement for protein synthesis during the process. These observations imply the presence of an active and inactive phenylalanine hydroxylase with the relative amounts of each determined by the cell density. The effects of db-cAMP are discussed. Evidence is presented here that the hydrocortisone stimulation of phenylalanine hydroxylase activity works through a different mechanism than the cell density dependent process.     

The existence of an inactive form of transglutaminase within metastasising tumours

Separation by anion exchange chromatography of detergent extracts from a poorly metastatic HSV-2-induced hamster fibrosarcoma, its highly metastatic variant and a highly metastatic rat fibrosarcoma indicated the presence of an inactive form of transglutaminase antigen, when eluent fractions were assayed for transglutaminase activity and antigen. This inactive antigenic transglutaminase was clearly separable from the particulate and cytosolic forms of the transglutaminase enzyme. Unlike tumours, its presence could not be demonstrated in extracts from normal rat liver. Measurement of activity levels during tumour growth indicated that the progression of the two highly metastatic tumours was accompanied by a decrease in cytosolic transglutaminase activity, whilst the activity of this enzyme form remained constant in the poorly metastatic tumour. Measurement of antigen levels indicated an inverse relationship between the level of inactive transglutaminase and the level of cytosolic transglutaminase activity, suggesting that the two forms are inter-related. Gel filtration indicated the molecular weight of the inactive form to be greater than both the particulate and cytosolic forms, and it was estimated to be 120,000. Partial proteolysis of the semi-purified inactive form, by either trypsin or thrombin, led to its activation and to the appearance of a transglutaminase similar in molecular weight and ionic mobility, both by anion-exchange chromatography and electrophoresis, to the cytosolic transglutaminase.     

Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme

We have studied the growth on acetate, the metabolism of acetate enzymes, and respiration of a series of citrate synthase mutants of Saccharomyces cerevisiae. The results confirmed and extended our previous observation that cytosolic citrate synthase is not necessary for growth on acetate. Deletion of mitochondrial citrate synthase (CS1) protein resulted in changes in metabolites, decrease in the amounts of pyruvate and alpha-ketoglutarate dehydrogenase complexes, reduced mitochondrial respiration of citrate and isocitrate, and an inability to grow on acetate. Using site-directed mutagensis, we constructed two separate CS1 proteins with mutations in the enzyme's active site. The mitochondria of cells carrying either site-directed mutagenized CS1 contained the inactive citrate synthase protein. With one mutant in which His313 was replaced with a glycine (CS1/H313G), growth on acetate was restored, and mitochondrial respiration of citrate and isocitrate increased toward parental levels as did the levels of several enzymes. With the other mutant CS1 in which Asp414 was replaced with a glycine (CS1/D414G), no growth on acetate or changes in other parameters was observed. We propose that the characteristics of the strain carrying the CS1 with a H313G mutation result from the formation of an intact Krebs cycle complex by the inactive but structurally unchanged H313G protein.