Microbial fermentative preparation of L-[15N2]lysine and its tracer: application to serum amino acid kinetic studies

The microorganism Brevibacterium flavum 21129 has been used to produce multigram batches of L-[15N2]lysine of high purity and isotopic enrichment by supplementation of the growth medium with (15NH4)2SO4 of 98.0 atom% excess. The doubly 15N-labeled lysine can be detected at dilutions 10 times greater than singly labeled lysine when isotope dilution curves are analyzed by gas chromatography-mass spectrometry. This enhanced sensitivity permits kinetic measurements of plasma free-lysine isotope content over a 300-fold dilution during 6 h following a single oral bolus of 5 mg/kg body wt. This inexpensive preparation method lends itself to the production of highly useful biochemical compounds for kinetic studies of human nutrition.     

Ion-pair extraction of bile acids with Lipidex gel

A new method for the extraction of bile acids from aqueous solutions, urine, plasma, and bile is described. A buffered solution of decyltrimethylammonium bromide is added to the sample to give a 0.03 m concentration of the counter-ion. The mixture is passed through a bed of Lipidex 1000, which is then washed with the buffered solution of counter-ion followed by water. The decyltrimethylammonium salts of bile acids are sorbed by the Lipidex and are eluted with methanol. Recoveries of unconjugated, taurine- and glycine-conjugated, sulfated, and glucuronidated bile acids are close to 100%. Unconjugated bile acids can also be quantitatively extracted from aqueous solutions and urine after acidification with acetic acid.     

Determination of copper concentration in blood plasma and in ocular and cerebrospinal fluids using graphite furnace atomic absorption spectroscopy

Published values for the concentration of Cu in cerebrospinal and intraocular fluids cover a very wide range (0.016 to 1.0 microgram/ml) and include values which are several times higher than those which would be consistent with normal physiology. An atomic absorption spectrophotometer equipped with a graphite furnace was used to measure the Cu concentration in these fluids and in blood plasma of toads, rabbits, and cats. Under standard conditions, these fluids yielded high background absorbance and only fractional recovery of added Cu. Parameters were therefore established which eliminated both the high background and the matrix interference and allowed the determination of Cu in 10-microliters aliquots of diluted blood plasma and undiluted cerebrospinal and ocular fluid samples. Under these conditions the Cu measured in the ocular (0.011 to 0.032 microgram/ml) and cerebrospinal fluids (0.033 to 0.050 microgram/ml) of these three species was lower than most previously reported values and only a small fraction (1-3%) of the concentration of Cu in the plasma of the same animals (0.85 to 1.22 micrograms/ml).     

Quantitative determination of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography

A method to separate the four major bases (cytosine, guanine, thymine and adenine) and the two minor modified bases (5-methylcytosine and 6N-methyladenine) in DNA has been developed. For optimal separation, several different buffer systems are available for isocratic elution. The 12 5-methylcytosine (5-mC) residues in the plasmid pBR322 can be determined with a deviation of less than 3% of the expected value and have been used for internal standardization. Formic acid hydrolysis of bases and probably of DNA does not lead to the deamination of cytosine or 5-mC and thus can be used routinely for DNA hydrolysis. Adenovirus or baculovirus DNA does not contain detectable amounts of 5-mC. The distribution of 5-mC in hamster cell DNA appears to be nonrandom in that different 5'-CpG-3'-containing restriction sites are methylated to different extents.     

Purification from Synaptosomal Plasma Membranes of Calpain I, a Thiol Protease Activated by Micromolar Calcium Concentrations

Synaptosomal plasma membranes (SPMs) were prepared from whole rat brain and assayed for calcium-stimulated proteolytic activity. Addition of calcium to SPMs caused a dose-dependent increase in trichloroacetic acid-soluble protein. Two peaks of protease activity directed against a casein substrate were detectable when SPMs were incubated with low-ionic-strength buffer and the extract was fractionated on DEAE-cellulose. The enzyme in peak 1 required less than 1/10 the calcium concentration for activation as the peak 2 protease (Kact1 = 35 microM; Kact2 = 500 microM). The specific thiol-protease inhibitors leupeptin and antipain and the alkylator iodoacetate blocked enzyme activity. The low-sensitivity protease was converted to a high-sensitivity enzyme (Kact = 20 microM) by substrate affinity chromatography in the presence of calcium. This protease was purified 550-fold from SPMs. The high- and low-sensitivity membrane-associated calcium-dependent proteases are part of a family of enzymes, the calpains, previously reported in cytosolic fractions of several tissues.     

Evidence that cyclic AMP may regulate Ca2+-mobilization and phospholipases in thrombin-stimulated human platelets

The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca²⁺ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca²⁺ ionophore, appeared to parallel the decrease in Ca²⁺ uptake activity. Phospholipase A₂ activity, monitored by the production of [³H]lysophosphatidylcholine and [³H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca²⁺ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca²⁺ content.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae

The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form). This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short form protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similar to wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form of the enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminal amino acids of hexokinase II are not required in vivo either for phosphorylation of hexoses or for glucose repression.     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.