Peak Specific Activity Research Articles

Purification and some properties of two forms of chitinase from mycelial cells of Mucor rouxii.

Chitinase activity was measured in extracts of mycelial cells of Mucor rouxii as a function of the culture age. There was a peak of specific activity at the mid-exponential phase of growth (10 h), which paralleled chitin synthase activity. An additional peak of chitinase with higher specific activity was detected in 4 h cultures, which coincided with the onset of germination. Purification of chitinase activities from the cytoplasm revealed two enzymes, I and II, with different molecular mass and ionic charge. Antibodies induced with chitinase I did not cross-react with chitinase II. Both enzymes digested nascent chitin preferentially over preformed chitin, yielding diacetylchitobiose as the sole product of hydrolysis.

Role of the carbon source in regulating chloramphenicol production by Streptomyces venezuelae: studies in batch and continuous cultures.

Both carbon- and nitrogen-limited media that supported a biphasic pattern of growth and chloramphenicol biosynthesis were devised for batch cultures of Streptomyces venezuelae. Where onset of the idiophase was associated with nitrogen depletion, a sharp peak of arylamine synthetase activity coincided with the onset of antibiotic production. The specific activity of the enzyme was highest when the carbon source in the medium was also near depletion at the trophophase-idiophase boundary. In media providing a substantial excess of carbon source through the idiophase, the peak specific activity was reduced by 75%, although the timing of enzyme synthesis was unaltered. Moreover, chemostat cultures in which the growth rate was limited by the glucose concentration in the input medium failed to show a decrease in specific production of chloramphenicol as the steady-state intracellular glucose concentration was increased. The results suggest that a form of "carbon catabolite repression" regulates synthesis of chloramphenicol biosynthetic enzymes during a trophophase-idiophase transition induced by nitrogen starvation. However, this regulatory mechanism does not establish the timing of antibiotic biosynthesis and does not function during nitrogen-sufficient growth in the presence of excess glucose.

Evidence for nucleoside channeling in vivo: Deoxythymidine incorporation into rat liver dTTP and nuclear matrix DNA

Previous studies in prokaryotes and in eukaryotic cell lines have indicated the possible existence of more than one dTTP pool accessible to DNA synthesis. To investigate this possibility in eukaryotes invivo, the incorporation of [3H]deoxythymidine into nuclear matrix-attached DNA and intracellular dTTP was examined in regenerating rat liver. The labeling of matrix DNA reached a maximum after a 5 min pulse and then began to rapidly decrease. Conversely, [3H]deoxythymidine incorporation into dTTP began to increase after 5 min and peaked 10 min after injection. Since the peak specific activity for [3H]deoxythymidine incorporation into matrix DNA precedes that into dTTP, there seems to be channeling of exogenous thymidine directly to sites of DNA replication, bypassing existing nucleotide pools.

Nutrition and Exocrine Pancreatic Function in Infancy

Thirty-six infants suffering from gastrointestinal diseases during infancy were evaluated for exocrine pancreatic function by means of the pancreozymin-secretin test. Duodenal fluid volume, protein content, and content and peak-specific activity of amylase, lipase, and trypsin were determined following intravenous administration of either pancreozymin (CCK) or secretin. Seven infants receiving long-term parenteral nutrition and 17 infants receiving oral feedings and exhibiting first-degree malnutrition in association with chronic diarrhea underwent testing. Results were compared to those of a group of 12 age-matched infants with chronic diarrhea and weight loss. Four patients receiving total parenteral nutrition (TPN) suffered from severe short bowel syndrome and three had intractable diarrhea of infancy. The total duration of TPN ranged from 1.5-12 months, and the period of exclusive TPN in the absence of oral feedings ranged from 1-4 months. The heights, weights, and weight-for-height relationships were at or above the fifth percentile for all seven infants. The weight-for-height relationship of each patient in the control group was above the fifth percentile, while that of all malnourished infants was below the fifth percentile. No significant difference was found in the volume of fluid collected following either CCK or secretin administration. The content and concentration of amylase and lipase were lower in those patients receiving TPN. The levels were statistically significant (p less than 0.05) following secretin administration. In contrast, the peak-specific activity and total trypsin content, as well as protein content, was not significantly different in patients receiving TPN, controls, and malnourished patients.(ABSTRACT TRUNCATED AT 250 WORDS)

TOTAL PARENTKRAL NUTRITION (TPN) IN INFANCY AFFECTS AMYLASE AND LIPASE BUT NOT TRYPSIN SECRETION

During prolonged fasting, the absence of enteric stimulation might lead to pancreatic hypofunction or atrophy. The ahility of the pancreas to respond to exogenous secretagogues following fasting in conjunction with TPN in infancy is not known. In addition, while severe degrees of malnutrition (M) are known to adversely affect pancreatic structure and function (PF), it is unknown whether infants presenting with chronic diarrhea and first degree M could have alterations in PF. The results of panereozymin-secretin tests were analyzed in the following patient groups: 7 infants receiving TPN for >6 weeks for severe diarrhea in association with short bowel syndrome or intractable diarrhea; 17 infants exhibiting first degree M and chronic diarrhea and 12 controls (age matched well nourished infants with chronic diarrhea). No significant difference was found among the 3 groups in the volume of fluid collected following either cholecystokinin or secretin. The content and concentration of amylase and lipase were lower in those receiving TPN following secretin (p<0.05) .Peak specific activity and total trypsin and protein content were similar in all groups The findings indicate that fasted infants receiving TPN retain their ability to respond to pancreatic secretagogues. However, the enzyme output is affected differently. Amylase and lipase exhibit an attenuated response whereas that of trypsin is maintained. No impairment in exocrine pancreatic secretion was found in infants suffering mild M.

Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities.

A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed 'dual-digitonin-pulse perfusion'. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.

Genetic and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2).

Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA 'complemented' several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin: S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (greater than 5 MDal) and a 49 kDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of 'secondary metabolites' whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the 'late exponential' phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to 'secondary' metabolism in general.

Betaine Stimulation of Vitamin B12 Biosynthesis in Pseudomonas denitrificans May Be Mediated by an Increase in Activity of -Aminolaevulinic Acid Synthase

The stimulation by betaine of vitamin B12 and extracellular coproporphyrin production in the industrial species Pseudomonas denitrificans GY57/2 was confirmed using a replacement culture technique; biosynthesis of haem was also found to be stimulated. In the absence of betaine, δ-aminolaevulinic acid synthase decreased in specific activity from time zero. In the presence of betaine, the enzyme activity increased markedly after 3 h and then decreased after exhaustion of extracellular betaine. The peak specific activity of δ-aminolaevulinic acid synthase increased in a dose-dependent manner. The enzyme was inhibited by haem. δ-Aminolaevulinic acid dehydratase was present in considerable excess over δ-aminolaevulinic acid synthase under all conditions studied and its specific activity was not affected by the addition of betaine. Our tentative conclusion is that the betaine effect is exerted via some positive effect on δ-aminolaevulinic acid synthase, the initial, rate-limiting and very labile enzyme of porphyrin and corrin biosynthesis.

Incorporation of radioactive phospholipid into subclasses of high-density lipoproteins

The incorporation of orally administered phospholipid into plasma high-density lipoproteins (HDL) was studied in three subjects. Plasma was analyzed by equilibrium density gradient ultracentrifugation, 5, 6, and 8 h after ingestion of 1.1 g [3H-choline, 14C-dilinoleoyl]phosphatidylcholine. At all time points in all subjects, there was a peak of phosphatidylcholine specific activity in fractions of density approximately 1.10-1.13 g/ml, corresponding to the subclass previously designated HDL2a. There was also a more variable, smaller peak of specific activity of phospholipids in HDL2b (1.063-1.100 g/ml) and in fractions of density approximately 1.19 g/ml. In the 1.10-1.13 fraction, 97 and 71%, respectively, of the 3H and 14C radioactivity were in phospholipids. The 3H/14C ratio was similar in phospholipids of HDL subfractions, the d less than 1.07 fraction, and in the administered phospholipid. The results show preferential transfer or exchange or absorbed phosphatidylcholine into specific subclasses of HDL.

A purification of microsomal glucose-6-phosphatase from human tissue

Glucose-6-phosphatase, an enzyme of the microsomal fraction, is a major intermediate in the mobilization of glucose from liver cells. Lack of a purified preparation of this enzyme has hampered efforts to understand the molecular details of Glycogen Storage Disease Type Ia (von Gierke's disease). The present study was undertaken to purify this membrane-bound enzyme from human placenta and liver using Sepharose affinity chromatography with glucose-6-phosphate as the bound ligand. Of the two tissues tested, the placenta gave the better results, perhaps because the purification began with fresh tissue. Protein eluting from the affinity column for a placental preparation gave three peaks of specific activity representing 45-, 33-, and 600-fold purification with a yield of about 2%. Specific activity determined for liver tissue was far more variable and represented a purification of about 5-fold. SDS-PAGE of protein from both tissues indicated only three bands in the range of 58–64,000 molecular weight. Although not purified to homogeneity, the scheme reported here represents a significant advance in the purification of functional G6Pase from human sources.

Thymidylate synthetase during synchronous nuclear division cycle and differentiation of Physarum polycephalum

Thymidylate synthetase and thymidine kinase activities in wild type strain M 3b and in thymidine kinase-deficient mutant TU63 of Physarum polycephalum are studied. Whenever nuclear division occurs in macroplasmodia of wild type, thymidine kinase and thymidylate synthetase activities sharply increase, although the increase of thymidylate synthetase activity is less pronounced than thymidine kinase activity. This is also true for other investigated nuclear divisions during the life cycle of P. polycephalum. It is shown for the first time that thymidylate synthetase is a periodically fluctuating enzyme during the naturally synchronous nuclear division cycle of P. polycephalum with a peak of specific activity in the S phase. In macroplasmodia, as well as after germination of microsclerotia of M 3b, thymidine kinase is the dominant enzyme, whereas at the time of the precleavage mitosis in sporulating macroplasmodia thymidylate synthetase is the predominant enzyme. This study describes and compares both dTMP-synthesizing enzymes during proliferation and differentiation of the same organism.

Different kinetic fates of apolipoproteins A-I and A-II from lymph chylomicra of nonhuman primates. Effect of saturated versus polyunsaturated dietary fat.

Monkeys fed polyunsaturated fat had significantly lower plasma cholesterol (186 +/- 18 vs. 276 +/- 31 mg/dl) and high density lipoprotein (HDL) mass concentrations (466 +/- 28 vs. 518 +/- 34 mg/dl) than did animals fed saturated fat. Plasma apoA-I concentrations also were significantly lower and apoA-II levels were generally, though not significantly, lower in the group fed polyunsaturated fat. In vivo reinjection studies, using thoracic duct lymph chylomicra labeled with (131)I and HDL labeled with (125)I, were done in order to study the mechanism of plasma HDL-lowering by polyunsaturated dietary fat. The peak specific activity (SA) of HDL apoA-I derived from (131)I-labeled chylomicra occurred at 3 hr after injection (172 +/- 11% of 1 min S.A.) and then an exponential decay occurred indicative of a precursor-product relationship between chylomicron apoA-I and HDL apoA-I. In contrast, HDL apoA-II derived from (131)I-labeled chylomicra had no early S.A. increase and began to die away immediately after injection. Labeled apoA-I from chylomicron and HDL origin had similar plasma fractional catabolic rates (FCR = 0.34-0.38 vs. 0.32-0.38 d(-1), respectively); apoA-II from chylomicron or HDL origin also had similar FCR (0.46-0.51 vs. 0.42-0.51 d(-1), respectively), which were significantly shorter than those for HDL apoA-I. There was a consistent trend toward a higher FCR for HDL apoA-I or A-II of polyunsaturated fat-fed recipients. Chylomicron apoA-I/triglyceride and apoA-II/triglyceride mass ratios were lower in polyunsaturated fat-fed animals (A-I/TG = 1.56 x 10(-3); A-II/TG = 1.47 x 10(-3)) vs. saturated fat-fed animals (A-I/TG = 2.58 x 10(-3); A-II/TG = 2.77 x 10(-3)). It was concluded that: (1) dietary polyunsaturated fat significantly lowered plasma cholesterol, HDL, and apoA-I concentrations relative to saturated fat; (2) the HDL-lowering effect of the dietary polyunsaturated fat may be due to the combined effects of decreased apoprotein production by the intestine and increased HDL catabolism; and (3) in the blood, chylomicron apoA-I and A-II differ in their metabolic fates.-Parks, J. S., and L. L. Rudel. Different kinetic fates of apolipoproteins A-I and A-II from lymph chylomicra of nonhuman primates. Effect of saturated versus polyunsaturated dietary fat.

Measurement of bile acid production in hyperlipidemic man: does phenotype or methodology make the difference?

Bile acid production has been measured in 13 studies in 10 hyperlipidemic subjects by simultaneous use of isotope dilution kinetics and chemical balance methodology. When the data of all 13 studies were averaged, the correlation between the two sets of values was negative and weak (r - 0.01, NS). However, when the correlations for normoglyceridemic and hyperglyceridemic subjects were examined separately, a strong positive correlation was found between the values obtained by the two methods in normoglyceridemic subjects (r + 0.92, P less than 0.01) but not in hyperglyceridemic subjects (r - 0.25, NS). Examination of bile acid specific activity decay characteristics in eight studies, where bile was sampled up to six times within the first 24 hr after radio-labeled bile acid infusion, revealed differences in three rate of attainment of peak specific activity and in the time taken subsequently to achieve first order kinetic decay. However, analysis of the data from these eight studies by input-output analysis yielded values for primary bile acid synthesis no different that those generated by conventional isotope dilution kinetics. Thus, bile acid production in normoglyceridemic subjects may be accurately quantitated by either isotope dilution or chemical balance methodology. Our data, as well as results from other laboratories, indicate that the values obtained are strictly comparable. On the other hand, the quantitation of bile acid production in hyperglyceridemic subjects by isotope dilution kinetics gives higher values than those obtained by chemical balance methodology, and in addition, higher values than those obtained in patients with normal plasma triglyceride levels.

Flow kinetics of mouse histocompatibility antigens.

Major histocompatibility antigens of the mouse (H-2 antigens) are found on a variety of different cell types and constitute a class of integral membrane-bound glycoproteins involved in tissue graft rejection and immune surveillance. We used monospecific alloantibodies directed against mouse H-2 antigens and standard pulse-chase technique to investigate the flow kinetics of delivery of newly synthesized membrane constituents to the cell surface. [35S]Methionine was injected intraperitoneally, and, after a chase with unlabeled methionine, livers were excised and fractionated into endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions. Label first appeared in H-2 antigens located within the endoplasmic reticulum. Maximum specific activity observed between 5-7 min after injection of label was followed by a rapid loss of label. H-2 antigens of Golgi apparatus also were labeled early. Peak specific activity observed 15-25 min after injection of label was again followed by rapid loss of label. H-2 antigens of the plasma membrane were labeled last and appeared to accumulate radioactivity with no evidence of rapid turnover. These results provide evidence for a precursor-product relationship between H-2 antigens located within the cell on membranes of endoplasmic reticulum and Golgi apparatus and those on the plasma membrane. Flow of individual membrane-bound glycoproteins from their sites of synthesis and insertion into the membrane of the endoplasmic reticulum to the plasma membrane through the Golgi apparatus is indicated.

Metabolism of phosphatidylcholine in the frog retina

The biosynthesis and the turnover of phosphatidylcholine were studied in the frog retina following either (a) injection into the animal of 32PO 4, 33PO 4, [1,3- 3H]glycerol, [2- 3H]glycerol, or [ methyl- 3H]choline, or (b) incubation of isolated retinas in solutions containing [ methyl- 3H]choline. 1. 1. Examination of the pools of lipid precursors in the retina demonstrated that the choline and phosphate pools are long-lived compared to the glycerol pool, which is metabolically very active and turns over rapidly. 2. 2. The peak in specific activity of phosphatidylcholine synthesized from labeled glycerol occurred earlier, and was higher in the microsomal fraction than in the rod outer segments, which is consistent with synthesis of phosphatidylcholine on the microsomes of the inner segment and subsequent incorporation into the rod outer segments. 3. 3. Autoradiography of retinas incubated in vitro with tritiated choline revealed a diffuse labeling pattern in the rod outer segments. Biochemical studies following injection of labeled glycerol showed an exponential decline in specific radioactivity of phosphatidylcholine in the rod outer segments, which is consistent with a diffuse labeling of these membranes. 4. 4. The half-life of phosphatidylcholine in the rod outer segments synthesized from labeled glycerol was found to be 18–19 days. Based on these values, calculations were made which indicated that phosphatidylcholine in the outer segments is turning over faster than integral disc membrane proteins.

The action of lung lysosomal phospholipases on dipalmitoyl phosphatidylcholine and its significance for the synthesis of pulmonary surfactant.

Subcellular fractions were prepared from rabbit lung and characterized by marker enzyme assays. Phospholipases A of lysosomal type (activity at pH 4.0 in the absence of Ca2+) show a peak of specific activity in fraction P3 (13000 x g; 20 min; pellet). About 70% of the phospholipase A activity of P3 can be obtained in a soluble extract. The rates of hydrolysis of dipalmitoyl (saturated) and unsaturated phosphatidylcholine molecules, presented to enzyme preparations in liposomes of varying composition, are shown in Tables 3 to 5. Although the rate of unsaturated molecules is unaffected by liposome composition, saturated molecules are hydrolyzed significantly faster in 5 mol % dipalmitoyl phosphatidylcholine liposomes than in 95 mol %. With enzyme preparation I (60% A1: 40% A2) the increase was 6-fold (P less than 0.01). Preparations I and II hydrolyzed both kinds of molecules at similar rates in 50 mol % dipalmitoyl phosphatidylcholine liposomes, but preparation III, with a higher proportion of A2 (80%), gave a notably lower rate of hydrolysis of saturated molecules. Phosphatidylglycerol (10 mol %) also decreased the rate of hydrolysis of saturated molecules. These results are discussed in relation to fluidity of the liposomes and to the functions of the lysosomal-type phospholipases A of the lamellar inclusion bodies of the lung.

Keratins in cultivated human keratinocytes are stable.

Cultures of human keratinocytes were established according to the technique of Rheinwald and Green. When cultures were exposed to [14C] leucine, the uptake of leucine and increase in the specific activity of 6 urea-extractable polypeptides was prompt-each keratin achieved 50% of its peak specific activity in 3-6 hours. Cultures were exposed to [14C] leucine for 6 hours and then permitted to grow in unlabeled medium for 10 days. These confluent cultures shed cells into the medium; the amount or protein shed daily was 22.2 microgram or roughly 0.9% of the protein of the attached cells. Thus, protein shed into the medium over a 10-day period of the pulse-chase experiment was 9% of the total extractable protein. The specific activity of individual polypeptides extracted by urea fell an average of 25% during the 10-day chase. Polypeptides extracted by buffer A showed a fall in specific activity of 55% over this period. The relative amounts of individual urea-extractable polypeptides and individual buffer A-extractable polypeptides remained constant over a 10-day period. The rapid labelling of all urea-extractable polypeptides and the relative stability in the specific activity of these polypeptides is evidence that one keratin is not modified to form another keratin and that, once synthesized, the molecules are stable.

Subcellular distribution of intraportally injected 125I-labeled insulin in rat liver

Time course studies revealed that at 30 s after intraportal injection of 200 μU of 125I-labeled insulin per 100 g rat 47.9 ± 2.8% of the injected radioactivity was recovered from the liver homogenate by precipitation with trichloroacetic acid. Trichloroacetic acid precipitable radioactivity declined to very low levels during the next 30 min whereas trichloroacetic acid soluble radioactivity reached a peak value of 9.56 ± 1.9% at 5 min and declined gradually thereafter. At 30 s mean peak accumulations ±SE of 6.83 ± 0.42, 5.06 ± 0.27, 14.90 ± 1.85, and 3.58 ± 0.58% of injected radioactivity were recovered in trichloroacetic acid precipitates from the 700 g (nuclei + debris), 10,000 g (mitochondria + lysosome), 105,000 g (microsomes), and supernatant (cytosol) subfractions, respectively. Mean peak values of 0.72 ± 0.08, 0.12 ± 0.02, and 1.11 ± 0.16% of injected radioactivity were recovered in the partially purified mitochondrial fraction, purified nuclei, and plasma membranes, respectively, as trichloroacetic acid precipitable material. Most of the trichloroacetic acid precipitable activities in the subfractions were immunoprecipitable. Trichloroacetic acid soluble radioactivity was found mainly in the cytosol and microsomal fractions. Peak specific activity (percentage of injected dose/mg protein × 10 −3) was highest in the microsomes, intermediate in the plasma membranes, and very low in the purified nuclei and partially purified mitochondrial fraction. The specific activity of the microsomes remained at or near peak levels for 5 min after 125I-labeled insulin injection and then declined, whereas specific activity of the plasma membranes dropped precipitously to 25% of peak values at 5 min. Sephadex gel filtration of the radioactivity in the deoxycholate soluble fraction of microsomes at 5 min after 125I-labeled insulin injection resulted in the elution of a major peak (Peak I) in the region of 125I-labeled insulin and a minor peak (Peak II) in the region of the labeled A and B chains. Incubation of the fraction for 30 min at 37 °C with 3 m m reduced glutathione and 15 m m EDTA resulted in a reciprocal fall in Peak I and rise in Peak II. The data suggest that intraportally injected 125I-labeled insulin is rapidly internalized and concentrated in the rat liver microsomes. The time courses of appearance and disappearance of trichloroacetic acid precipitable radioactivity in plasma membrane and microsomes further suggest, although do not prove, that insulin binds to plasma membranes before it is internalized. They also provide presumptive evidence suggesting that the sequential degradative pathway is operative in vivo.

Metabolic Fate of Chylomicron Phospholipids and Apoproteins in the Rat

To study the metabolic fate of chylomicron phospholipid and apoproteins, 15 mg of doubly labeled ([(3)H]leu, [(32)P]phospholipid) rat mesenteric lymph chylomicrons were injected as an intravenous bolus into conscious rats. The specific radioactivity, composition, pool size, and morphology of the plasma lipoproteins were determined after 2-60 min. After injection of chylomicrons, there was a rapid transfer of radioactivity into high density lipoproteins (HDL). At peak specific activity in HDL (2-5 min), 35% of injected apoprotein and 25% of phospholipid radioactivity were recovered in HDL (d 1.063-1.21 g/ml), with smaller recoveries in other lipoproteins and liver. There was an initial rapid rise of (32)P specific activity in HDL and d 1.02-1.063 lipoproteins (low density lipoproteins [LDL]), but whereas LDL specific activity subsequently converged with that of d < 1.02 lipoproteins, HDL specific activity decayed more rapidly than LDL or d < 1.02 lipoproteins. Lipolysis of chylomicrons was associated with a transfer of phospholipid mass into LDL and HDL. At 5 min, 80% of injected triglyceride had been lipolyzed and there was a significant increase in phospholipid mass in LDL and a smaller increase in HDL. At 10 min, the mass of phospholipid in LDL had returned towards control values, and there was a further increase in phospholipid mass in HDL, which suggested phospholipid transfer from LDL to HDL. In donor lymph chylomicrons (3)H-radioactivity was present in apoprotein (apo)B, apoA-I, and apoA-IV, but only radioactivity of apoA-I and apoA-IV were transferred to HDL. Transfer of radioactivity was associated with loss of mass of apoA-I and apoA-IV from the fraction that contained the chylomicron remnants (d < 1.02). With injection of 15 mg chylomicron, there was a small but insignificant increase in the relatively large pool of HDL apoA-I. However, 60 min after injection of 250 mg of human or rat intestinal chylomicrons into the rat, there was a significant increase in HDL apoA-I that resulted from acquisition of a major fraction of the chylomicron apoA-I. After injection of chylomicrons, phospholipid vesicles were observed by negative stain electron microscopy in the LDL and HDL ultracentrifugal fractions, especially in the LDL. Upon addition of an osmotically active compound, cellobiose, vesicles were observed as flattened particles with a double lipid bilayer thickness ( congruent with 100 A). To validate further the identity of these particles, chylomicrons were injected into rats with [(3)H]glucose, and the recipient rats' plasma was fractionated by chromatography on 6% agarose. Trapping of [(3)H]glucose occurred in the void and LDL regions of the column, and vesicular particles were identified in these column fractions by negative stain electron microscopy. Catabolism of chylomicrons is associated with a rapid transfer of phospholipid, apoA-I, and possibly apoA-IV into HDL. Chylomicron phospholipid appears to give rise to vesicles which are probably incorporated into preexisting HDL. Chylomicron surface components may be an important source of plasma HDL.

Alterations of nucleic acid and protein syntheses in vivo in the chick embryo mediated by captan.

1. The objective of this investigation was to measure the effects of captan on DNA, RNA and protein biosyntheses in limbs of developing chick embryo, in vivo. 2. Captan (12p.p.m.) was injected into the egg on day 4 of incubation and macromolecular syntheses were measured on days 8--14. 3. Total DNA content was unaffected by captan, but incorporation of [3H]thymidine was inhibited over the entire time range; the period of peak specific activity (day 11) was inhibited in treated samples to 67% of control. 4. Total RNA content was reduced in the earlier days but returned to normal by day 14, whereas incorporation of [3H]uridine was lowered throughout the period. The timing of peak specific activity for RNA synthesis was delayed by 2 days in treated eggs and was inhibited by 32% when peak days were compared. 5. Total protein concentration was slightly lowered by captan treatment in the mid-range of the days measured and the incorporation of [3H]valine was retarded in the early days; peak periods of synthesis were similar in magnitude but were shifted from days 9--10 in control to day 11 in treated embryos.