Biomedical subjects
Publications and source records attributed to P J Lad.
Four tandem subtilisin-like protease genes were found on a 6,854-bp DNA fragment cloned from the alkalophilic Bacillus sp. strain LG12. The two downstream genes (sprC and sprD) appear to be transcribed independently, while the two upstream genes (sprA and sprB) seem to be part of the same transcript.
Explore the source record for details and available documents.
1. Two new extracellular serine proteases have been purified to homogeneity from Streptomyces fradiae. 2. On amino acid sequencing, striking homology is observed between the first enzyme and Streptomyces griseus Protease A, and the second enzyme and S. griseus trypsin. 3. The sequence information shows for the first time that structurally and enzymatically related serine proteases are extracellularly expressed by different Streptomycetes. 4. Differential keratinolytic substrate specificity among these two microbes are probable due to a difference in disulfide reduction capacity.
Explore the source record for details and available documents.
Long-term primary adult rat hepatocyte cultures show growth-state-dependent changes in adenylate cyclase and cAMP phosphodiesterase activities. Cellular adenylate cyclase activity decreases to undetectable levels within 1 day postplating, reappears on Days 4-5, and becomes maximal on Day 9. Membrane adenylate cyclase and cellular cAMP formation are insensitive to glucagon during log phase (Days 4-8) but not during lag (Day 1) or stationary phase (Day 12). Cyclic AMP phosphodiesterase activities (soluble and particulate) fall approximately equal to 70% by Day 2 but recover as proliferation begins. By contrast, the particulate phosphodiesterase assayed at 100 microM cAMP, decreased during Days 0-2. These observations simulate changes seen during liver proliferative transitions in vivo and, therefore, further support the use of these cultures as a developmental model.
Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.
Hepatic alcohol dehydrogenase activity and mass content change coordinately during development in male rats. Enzyme activity and mass content increase continuously after birth to 100 and 80% of maximal values within 6 weeks (2.6 +/- 0.4 mumole/min/g liver and 92 +/- 20 micrograms/g liver), respectively. When expressed per milligram of soluble proteins, both parameters peak at 3 weeks (0.052 +/- 0.002 mumole/min/mg protein and 2.0 +/- 0.4 micrograms/mg protein) and then decrease gradually to plateau levels. These decreases probably arise from a "surge" in soluble liver protein levels that occurs after weaning. Similar developmental patterns also occur in female rats. These findings are the first quantitative measurements of this enzyme in developing animals.
Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.
Monospecific rabbit antibodies against purified Fischer-344 rat liver alcohol dehydrogenase were produced and used to develop an enzyme-linked immunoadsorbent assay for alcohol dehydrogenase. The assay is based upon the competitive inhibition of specific antibody binding to antigen (alcohol dehydrogenase adsorbed onto plastic microtiter plates) by soluble alcohol dehydrogenase (contained in unknown sample extracts or in known standard solutions). The amount of bound antibody is determined following incubation with peroxidase-linked second antibody (goat anti-rabbit IgG antibody-peroxidase conjugate) by colorimetric measurements of peroxidase activity at 490 nm in the presence of O-phenylenediamine. The assay is highly sensitive (it detects 10-1000 ng alcohol dehydrogenase/50 microliter) and it offers a precise (interexperimental variations in samples were less than 10%), rapid (6-8 h), and specific method for measurements of alcohol dehydrogenase in tissue homogenates or cultured hepatocytes. The assay was used to study changes in alcohol dehydrogenase levels during the growth cycle of cultured hepatocytes over a 2-week period and in rat liver homogenates after starving the animals for 72 h. In cultured hepatocytes, alcohol dehydrogenase activity and immunoassayable enzyme levels decreased coordinately during lag and early log phase, from 13.2 +/- 1.2 to 5.0 +/- 1.0 micrograms enzyme/mg protein, respectively. In mid-log phase, the enzyme levels were very low (1.3 +/- 0.4 micrograms enzyme/mg protein). During stationary phase, the levels (5.7 +/- 0.6 micrograms enzyme/mg protein) increased to 35% of the levels of freshly isolated hepatocytes (15.6 +/- 1.4 micrograms enzyme/mg protein). In starved animals, the enzyme levels decreased from 7.56 +/- 0.55 to 2.97 +/- 0.27 mg enzyme/liver. These changes also coincided with decreases in activity from 8.84 +/- 0.35 to 6.56 +/- 0.68 microM/min/liver.
Livers of Sprague-Dawley rats contain 30-100% more alcohol dehydrogenase activity than livers of Fischer-344 rats. When weight-matched rats from both strains are injected with the same dose of ethanol (1.2 g/kg), Sprague-Dawley rats achieve lower blood alcohol levels than Fischer-344 rats at all the time-points tested. Purified alcohol dehydrogenases from both strains of rats exhibit identical electrophoretic mobilities in SDS-polyacrylamide Section and in isoelectric focusing slab gels, pH optima, peptide maps, Km for ethanol, and capacities to bind monospecific rabbit antibodies. Quantitative differences in alcohol dehydrogenase activity between these strains of rats are due to differences in their liver alcohol dehydrogenase levels.
Hormonal effects on gluconeogenesis from lactate were studied during the growth cycle of adult rat parenchyma liver cells using a primary monolayer culture system previously described [25]. Basal and glucagon-stimulated gluconeogenic ability were found to decline rapidly during log phase, insulin-stimulated growth. A progressive recovery of gluconeogenesis activity was observed after cell division subsided. Rates of lactate-gluconeogenesis were found also to decline in the absence of prior insulin exposure. This decline was not as rapid as the loss observed in cells cultured with insulin. However, in insulin-deficient cultures gluconeogenesis was completely abolished after 12 days and did not reelevate with further incubation unless cells were washed and exposed to glucagon. Decreasing growth rates of insulin-supplemented cultures by decreasing serum concentrations resulted in comparatively higher gluconeogenic activity. The results presented here are consistent with previous observations of hepatic parenchymal expression of 'differentiated function' during cellular growth phases in culture (i.e., differentiated functions are generally lost during rapid growth and regained as cells become quiescent). The present study, however, presents unexpected effects of insulin on the apparent growth-state dependent gluconeogenic recovery. Our data imply that although insulin has long been known to inhibit gluconeogenesis, its presence in culture may facilitate long-term basal maintenance of gluconeogenic enzyme activity. Insulin also functions as a growth factor whose initial mitogenic effect correlates with decreased gluconeogenic function. These changes show no simple or predictive correlation with cyclic nucleotide metabolism.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.