Decreased transport of D-glucose and L-alanine across brush-border membrane vesicles from small intestine of rats treated with mitomycin C

To elucidate the mechanisms underlying the dysfunctions of intestinal absorption induced by antitumor drugs, the effect of pretreatment with mitomycin C on sodium gradient-dependent D-glucose and L-alanine transports was studied in rat brush-border membrane vesicles. 24, 48, 96, or 120 h following a single intravenous injection of mitomycin C, brush-border membrane vesicles were prepared from rat small-intestines. The uptake of D-glucose and L-alanine was shown to be Na+ gradient-dependent even in the case of vesicles obtained from mitomycin C-treated rats, but uptake rates measured at 15 s and magnitude of overshooting effect in uptake of both solutes were decreased in vesicles maximally from 48 h mitomycin C-treated rats. The rate of D-glucose uptake calculated at 15 s recovered to the control level in vesicles prepared at 96 h and 120 h after mitomycin C-treatment, indicating that the effect of mitomycin C on Na+ gradient-dependent D-glucose transport would be fully reversible. Tracer exchange experiments under Na+ and D-glucose equilibrated conditions indicated that the Na+/D-glucose transporters were similarly operative in the vesicles from control and 48 h mitomycin C-treated rats. Rates of 22Na+ uptake measured at 15 s in vesicles from 48 h mitomycin C-treated rats, however, were increased. The increased permeability to Na+ might bring about a more rapid dissipation of the Na+ gradient in these vesicles and this would secondarily cause the decrease in Na+-dependent D-glucose uptake in vesicles from mitomycin C-treated rats.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.     

Casein kinase II activities related to hyperphosphorylation of human papillomavirus type 16-E7 oncoprotein in epidermal keratinocytes

The present study describes hyper-phosphorylation of E7-oncoprotein of human papillomavirus (HPV) type 16 in epidermal keratinocytes. We found that highly phosphorylated E7-oncoprotein was present in epidermal keratinocytes but little in fibroblasts. The E7 oncoprotein contains serine residues (Ser-Ser-Glu-Glu-Glu) capable of being phosphorylated by casein kinase II (CK II). Extracts from various cell lines including human origins transformed by HPV 16 were examined for the casein kinase activity. The results showed that CK II activity was present at significantly high levels in keratinocytes but little or no detectable levels of the activity in human fibroblasts. These differential CK II activities in host cells may play a part in the differential transforming activity by E7-oncoprotein.     

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.     

Microdissection studies of the structural alterations induced in rat kidneys by experimental postischemic acute renal failure

A unique opportunity presented itself for a morphologic study of experimental unilateral acute renal failure (ARF) in male rats. The ARF had been induced in the rats by temporary occlusion (1h) of the left renal artery. Twenty-nine rats were divided into subsets as follows: 2-3 h, 24 h, 1 week, 2, 4, 8, and 12 weeks following release of occlusion. Microdissection showed a heterogeneous population of abnormally structured proximal tubules in which the regressive lesions of tubular necrosis were combined with the progressive reaction of repair. The lesions demonstrated are reminiscent of those which have been described in ARF in the human and in experimental animals. Many proximal tubules in the 2- to 3-hour subset presented 1-3 disruptive lesions (DLs) while greater numbers of proximal tubules from the 24-hour group presented 1-5 DLs. Many proximal tubules presented no DLs, but nearly all from the 24-hour subset (97-100%) displayed a squamate appearance which paralleled and was caused by acute tubular necrosis. At 1 week, a dilated pars recta was common, but by this time, the squamate pattern had disappeared. Many casts were present. At 2 weeks, many fewer casts were present in proximal tubules and none were seen at 4, 8 or 12 weeks. The nephrons, particularly the proximal tubules, presented a variety of structural alterations at 2, 4, 8 and 12 weeks. Changes of special interest include (1) the presence of swan-necks; (2) a distinctive squamate appearance of the proximal tubules in the animals killed at 24 h; (3) a spiral, curled appearance caused by differential hyperplasia in animals at 4, 8 and 12 weeks, and (4) a tendency for ischemic lesions to involve all layers of the renal cortex.     

Plasma membranes purified from myeloid leukemia cells before and after differentiation : I. Characterization of spectrin-like proteins and increased association of actin

Two-step sucrose density gradient centrifugation was used to isolate the plasma membrane of a myeloid leukemia cell line (Ml). Calspectin (or fodrin) was identified in the Triton-insoluble fraction from the plasma membrane, and the molecular size and actin- and calmodulin-binding activity were studied. During differentiation of this cell line, which accompanied the induction of cell motility and phagocytic activity, the membrane-bound actin increased dramatically, whereas calspectin increased only slightly. Therefore, calspectin does not appear to have a major function in the increased binding of actin filaments to the plasma membrane, a requirement for the induction of cell motility.     

Effects of site-directed mutagenesis of the surface residues Gln128 and Gln225 of thermolysin on its catalytic activity

Thermolysin is remarkably activated and stabilized by neutral salts with varying degrees depending on salt species, and particular surface residues are thought to be especially important in its activity and stability [Inouye, K. (1992) J. Biochem. 112, 335-340; Inouye, K. et al. (1998) Biochim. Biophys. Acta 1388, 209-214]. In this study, we examined the mutational effects of the surface residues of thermolysin. Gln128 and Gln225 were selected as the residues to be mutated because they are located on the surface loop and close to but not in the active site (23.5 and 15.8 A far from the active site zinc ion, respectively) and fully solvent accessible. Nine single mutants [Q128K (Gln128 is replaced with Lys), Q128E, Q128A, Q225K, Q225R, Q225E, Q225D, Q225A and Q225V] were constructed by site-directed mutagenesis. Mutational changes in catalytic activity were found only in the mutant thermolysins having a hydrophobic residue at the position 225 (Q225A and Q225V). In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA), the alkaline pK(a) value of Q225A is 8.48 +/- 0.04, being higher by 0.42 +/- 0.07 units than that of the wild-type thermolysin. The k(cat)/K(m) value of the wild-type enzyme is enhanced 14 times with 4 M NaCl, and those of Q225A and Q225V are enhanced 10 and 19 times, respectively. In the hydrolysis of a negatively charged substrate N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), unlike FAGLA, the initial velocities of Q225A and Q225V decreased to 30 and 50% of that of the wild-type enzyme, respectively. Their thermal stability is similar to that of the wild-type enzyme. These findings indicate that even a single mutation at the thermolysin surface induces changes in the electrostatic environment in the active site and affects the activity. Thus, site-directed mutagenesis of surface residues of thermolysin, including apparently thermodynamically unfavorable introduction of hydrophobic residues, should be explored to improve its activity and stability.