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The retention of degradation of insulin by isolated perfused liver have been examined. Noncyclically perfused livers from streptozotocin-diabetic rats retained 25% and degraded 10% of 125I-insulin administered as a 1-min pulse. On gel filtration (Sephadex G50F), the degradation products released into the vascular effluent eluted in the salt peak. During the 45-min interval after the end of the 125I-insulin infusion, 0.19% of the total dose was excreted in the bile. 60-90% of this material consisted of iodinated, low-molecular-weight degradation products. Inclusion of native insulin with the 125I-insulin in the pulse depressed both the retention and degradation of iodinated material; however, this reflected increased retention and degradation of the total insulin dose (125I-insulin plus native hormone). The log of the total amounts of insulin retained and degraded were linearly related to the log of the total amount of insulin infused at concentrations between 12.7 nM and 2.84 muM. Increasing the amount of native insulin in the infused pulse also depressed the total amount of iodinated material found in the bile and led to the appearance in the bile of intermediate-sized degradation products that did not simultaneously appear in the vascular effluent. Addition of high concentrations of glucagon to the infused 125I-insulin had no effect on the retention or degradation of the labeled hormone, or on the apparent size and amount of iodinated degradation products found in the vascular effluent or in the bile. Preinfusion of concanavalin A inhibited both 125I-insulin retention and degradation. A greater depression by concanavalin A of degradation than binding was also observed with isolated hepatocytes. In contrast to 125I-insulin, the retention and degradation of two iodinated insulin analogues of relative low biological potency, proinsulin and desalanyl-desasparaginyl insulin, were small. The amount of radioactivity appearing in the bile after infusion of these analogues was almost negligible. However, degradation products of these analogues that appeared in the bile and in the vascular effluent was qualitatively similar to those found after the infusion of 125I-insulin. Our findings suggest that the rapid initial uptake of 125I-insulin after its infusion into noncyclically perfused liver, as well as its subsequent degradation, behaves in a qualitatively similar fashion to the binding of 125I-insulin and its degradation by isolated rat hepatocytes. This uptake and the subsequent phase of degradation may be attributable to binding of insulin at specific recognition sites, preliminary to its transfer to a degradative site(s) presumed to be located inside the cell.
Insulin and glucagon degradation by rat kidney homogenates and subcellular fractions was examined under a variety of conditions including high and low substrate concentrations, at pH 4 and pH 7, with and without glutathione. At high insulin concentration (4.1 - 10(-5) M) insulin degradation by the homogenate was greatest at pH 4 but at low insulin concentration (1 - 10(-10) M) insulin degradation was greatest at pH 7. At either high or low glucagon concentration glucagon degradation by the homogenate was greatest at pH 7. Glutathione at pH 7 stimulated insulin degradation at high insulin concentrations and inhibited insulin degradation at low concentrations; Glucagon degradation at pH 7 was inhibited at both high and low concentrations of glucagon by glutathionemseparation of kidney into cortex and medulla prior to homogenation produced a pattern of insulin and glucagon degradation identical to the whole homogenate but glucagon degradation by the medulla was greater than by the cortex. Examination of degradation by subcellular fractions revealed that at high concentration at neutral pH most insulin was degraded by the 100 000 X g pellet but at low insulin concentrations over 90% of the activity was in the 100 000 X g supernatant; At pH 7, at both high and low concentrations, most glucagon-degrading activity was in the 100 000 X g pellet, although the cytosol also had activity; At pH 4 most degradation occurred in the lysosomal fractions. Separation into cortex and medulla again showed similar distribution of activity as the whole gland with the medulla having more glucagon-degrading activity than the cortex. With low insulin concentrations the cortex 100 000 X g supernatant had higher relative specific activities than the medulla supernatant. Examination of recoveries of enzyme activity revealed that the subcellular fractions consistently had markedly less insulin-degrading activity than the original homogenate. This loss of activity was only discernible when insulin degradation was performed at pH 7 at low substrate concentrations. Comparable losses of glucagon-degrading activity were not seen.
Examination of insulin and glucagon degradation by rat kidney subcellular fractions revealed that most degrading activity was localized to the 100 000 X g pellet and 100 000 X g supernatant fractions. Further characterization of the degrading activities of the 100 000 X g pellet and supernatant suggested that three types of enzymatic activity were present at neutral pH. From the cytosol an enzyme with characteristics of the insulin glucagon protease of skeletal muscle was purified. This enzyme appeared to be responsible for insulin degradation by the kidney at physiological insulin concentrations. This enzyme also contributed to glucagon degradation but was not the most active mechanism for this. In the 100 000 X g pellet at least two separate enzymatic activities were present. One of these had properties consistent with those described for glutathione insulin transhydrogenase and appeared to be responsible for insulin degradation at high insulin concentration. The other enzyme was associated with the brush border and had properties consistent with the brush border neutral protease. This enzyme appeared responsible for glucagon degradation at both low and high substrate concentrations. An apparent marked synergism between the 100 000 X g pellet and the 100 000 X g supernatant was noted for insulin degradation at physiological insulin concentrations. Pellet glucagon-degrading activity and soluble insulin-degrading activity were necessary for this. The mechanism was found to be limited insulin degradation by the soluble enzyme resulting in both trichloroacetic acid-precipitable trichloroacetic acid-soluble fragments followed by further degradtion of the fragments by the glucagon-degrading enzyme resulting in an additional increase in trichloroacetic acid-soluble products.
The kinetics of the thermal degradation of promethazine in an acidic medium under various conditions were investigated. The degradation of promethazine and the formation of some degradation products were studied under aerobic and anaerobic conditions. The influence of pH, metal ions such as copper(II) and iron (III), and antioxidants was investigated. In an oxygen-saturated medium, promethazine generally followed first-order kinetics. Increasing the pH increased the degradation rate to a limiting value at pH 5. Addition copper (II) increased the degradation rate over the whole process, while iron (III) caused an increase for only a short time. Ascorbic acid sometimes increased the degradation rate, while higher concentrations of hydroquinone also accelerated the degradation. Pyrosulfite did not have any influence. Under anaerobic conditions, promethazine degraded only in the presence of copper (II) and iorn (III) ions. As a result of the studies on the qualitative and quantitative aspects of the oxidation process, a mechanism for the oxidative degradation of promethazine is suggested. Promethazine 5-oxide and a number of degradation products without intact side chains are formed via a semiquinone free radical. The influence of several factors on the degradation process is discussed.
A method for determining tissue sites of plasma protein degradation is described as applied to studies of low density lipoprotein (LDL) catabolism in swine. The method is based on the fact that sucrose is not degraded by lysosomal enzymes and thus accumulates in lysosomes. [(14)C]Sucrose was activated with cyanuric chloride and covalently coupled to the LDL protein. Studies in cultured fibroblasts have established that the sucrose (14)C accumulates intracellularly in degradation products at a rate equal to the rate of degradation of (125)I-labeled LDL simultaneously measured. In vivo the fractional catabolic rate of [(14)C]sucrose-LDL was the same as that of (125)I-labeled LDL. (14)C-Labeled degradation products in all major tissues were determined 24 hours after injection of [(14)C]sucrose-LDL. About 75% of the LDL degraded (calculated from analysis of the plasma decay curve) was accounted for in the (14)C-labeled degradation products accumulated in the tissues examined; only 4% appeared in the urine. In three studies, 37.9, 39.6, and 37.8% of the LDL degraded was recovered in the liver. Results were similar at 48 hr (38.7 and 39.9% hepatic degradation), but urinary losses were then about 10% and about 4% was lost in bile. All extrahepatic tissues examined contained (14)C-labeled degradation products. The concentration was highest in the adrenal glands-2 to 5 times that in liver and 10 times that in the next most active tissues. In principle this approach should be applicable to studies of the tissue sites of degradation of any of the plasma proteins.
Thyrotropin-releasing hormone (TRH)-degrading activity was investigated in human cord, maternal, and euthyroid adult sera by measuring (a) the rate of disappearance of TRH and (b) the rate of formation of degradation products. The rate of TRH degradation in cord and maternal sera was 25-33% of that in euthyroid adult serum. Concomitantly, in cord and maternal sera, the rate of formation of proline, a major TRH degradation product in serum, was one-quarter to one-third that in euthyroid adult sera. The differences were highly significant (P less than 0.001). The decreased levels of TRH-degrading activity in cord and maternal sera cannot be explained by (a) the presence of a dialyzable inhibitor, (b) the absence of an activator of TRH degradation, or (c) a reversal of the degradation process. There was no difference in the types of radioactive degradation products formed by cord, maternal, and euthyroid adult sera. The low level of TRH-degrading activity and its possible relationship to high thyrotropin-stimulating hormone levels in cord serum suggest that TRH-degrading activity may be a factor to consider in investigations of the perinatal pituitary-thyroid axis, but further studies are needed to determine the role of serum degradation of TRH in regulating physiological levels of TRH.
Microcystins (MCs) significantly threaten the ecosystem and public health. Biodegradation has emerged as a promising technology for removing MCs. Many MCs-degrading bacteria have been identified, including an indigenous bacterium Sphingopyxis sp. YF1 that could degrade MC-LR and Adda completely. Herein, we gained insight into the MCs biodegradation mechanisms and evolutionary dynamics of MCs-degrading bacteria, and revealed the toxic risks of the MCs degradation products. The biochemical characteristics and genetic repertoires of strain YF1 were explored. A comparative genomic analysis was performed on strain YF1 and six other MCs-degrading bacteria to investigate their functions. The degradation products were investigated, and the toxicity of the intermediates was analyzed through rigorous theoretical calculation. Strain YF1 might be a novel species that exhibited versatile substrate utilization capabilities. Many common genes and metabolic pathways were identified, shedding light on shared functions and catabolism in the MCs-degrading bacteria. The crucial genes involved in MCs catabolism mechanisms, including mlr and paa gene clusters, were identified successfully. These functional genes might experience horizontal gene transfer events, suggesting the evolutionary dynamics of these MCs-degrading bacteria in ecology. Moreover, the degradation products for MCs and Adda were summarized, and we found most of the intermediates exhibited lower toxicity to different organisms than the parent compound. These findings systematically revealed the MCs catabolism mechanisms and evolutionary dynamics of MCs-degrading bacteria. Consequently, this research contributed to the advancement of green biodegradation technology in aquatic ecology, which might protect human health from MCs.
The ability of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 to degrade aflatoxin varied depending on the substrate used to grow the mold. Substrates which allowed substantial mycelial growth yielded mycelia which actively degraded aflatoxin. Substrates which allowed minimal growth of mycelia yielded mycelia with little ability to degrade aflatoxin. Biodegradation of aflatoxin was also strain-dependent. A. parasiticus NRRL 2999 and NRRL 3000 actively degraded aflatoxin, A. flavus NRRL 3353 was less active, and A. flavus NRRL 482 and A. parasiticus NRRL 3315 degraded minimal amounts of aflatoxins. Those aspergilli producing greatest amounts of aflatoxin also degraded aflatoxins most rapidly, whereas those strains which produced minimal amounts of aflatoxin generally degraded aflatoxins less effectively. Substrates which allowed maximum aflatoxin production also yielded mycelia which actively degraded aflatoxins, whereas media which allowed limited production of aflatoxin generally yielded mycelia with minimal ability to degrade the toxin. Although exceptions exist, generally as aflatoxin production increased so did the ability of mycelia to degrade the toxin.
1. Rates of degradation of normal and abnormal protein were measured in hepatoma cells after labelling first for 16h with [14C]leucine plus L-arginine and then for 3h with [3H]-leucine plus the arginine analogue, L-canavanine. 2. Over the first 2h of the degradation period, canavanine-containing proteins were degraded at approximately 5 times the average degradation rate of normal proteins. 3. Degradation of normal proteins was inhibited by about 30% by insulin, cycloheximide, puromycin, leupeptin, antipain and foetal calf serum, whereas these agents had a negligible effect on the breakdown of canavanine-containing proteins. 4. Other compounds inhibited degradation of both classes of protein to equal extents. 5. Combination experiments showed no additional inhibitory effects on the degradation of normal proteins over degradation measured in the presence of a single selective inhibitor. 6. In contrast with the results with a 16 h labelling period, the degradation of normal proteins labelled for only 3 h was not inhibited by insulin. 7. These results are explained by a model with two distinct pathways of protein turnover. The first of these pathways involves the formation of autophagic vacuoles and would be completely inhibited by each of the selective inhibitors. Normal and canavanine-containing proteins would be catabolized by this pathway at equal rates. We propose that degradation by a second pathway is not regulated by the agents tested, but by the inherent stability of each protein.
The autosomal dominant ABH secretor gene together with the ABO blood type gene control the presence and specificity of A, B, and H blood group antigens in human gut mucin glycoproteins. Certain obligate anaerobes in feces produce extracellular antigen-specific glycoside structures. We estimated the populations of these bacteria in feces of 22 healthy subjects by determining the greatest dilution of feces that yielded A, B, or H blood group-degrading enzyme activity after 24 h incubation in anaerobic cultures. Comparatively small populations of fecal bacteria produce blood group-degrading enzymes; their estimated populations were 10(8) per g or less in 21 subjects. Fecal populations of B-degrading bacteria were stable over time, and their population density averaged 50,000-fold greater in blood group B secretros than in other subjects. We present evidence that the greater fecal populations of B-degrading bacteria in B secretors is due in part to a competitive nutritional advantage gained by their ability to enzymatically cleave the B antigenic determinant alpha-D-galactose from gut mucins of B secretors. Fecal populations of bacteria producing A and H antigen-degrading enzyme activities were comparable in all subjects to the fecal population of B-degrading bacteria in B secretors. The large populations of fecal anaerobes may be an additional source of A antigen substrate for A-degrading bacteria; thus, antigens cross-reacting with A antigen were detected on cell walls of anaerobic bacteria from 3 of 10 cultures inoculated with 10(-10) g feces. Bacteria producing B-degrading activity likely represent a separate population from those producing A- or H-degrading activity since their fecal populations differed numerically in 14 subjects. These findings suggest that adaptation of blood group-degrading enzymes to mucin structures in human colon ecosystems is chiefly by mutation-selection of comparatively small populations of constitutive enzyme-producing strains rather than by substrate induced enzyme synthesis in many strains.