Supernatant fraction

Zinc. The 2—3 g of zinc in the human body are widely distributed in every tissue and tissue duid (90—92). About 90 wt % is in muscle and bone unusually high concentrations are in the choroid of the eye and in the prostate gland (93). Almost all of the zinc in the blood is associated with carbonic anhydrase in the erythrocytes (94). Zinc is concentrated in nucleic acids (90), and found in the nuclear, mitochondrial, and supernatant fractions of all cells. 

Plasma Add cold acetonitrile, centrifuge, separate supernatant, fractionate by reverse-phase HPLC and TLC GC/MS and PNMR No data No data Weiss etal. 1994... 

Indicine IV-oxide (169) (Scheme 36) is a clinically important pyrrolizidine alkaloid being used in the treatment of neoplasms. The compound is an attractive drug candidate because it does not have the acute toxicity observed in other pyrrolizidine alkaloids. Indicine IV-oxide apparently demonstrates increased biological activity and toxicity after reduction to the tertiary amine. Duffel and Gillespie (90) demonstrated that horseradish peroxidase catalyzes the reduction of indicine IV-oxide to indicine in an anaerobic reaction requiring a reduced pyridine nucleotide (either NADH or NADPH) and a flavin coenzyme (FMN or FAD). Rat liver microsomes and the 100,000 x g supernatant fraction also catalyze the reduction of the IV-oxide, and cofactor requirements and inhibition characteristics with these enzyme systems are similar to those exhibited by horseradish peroxidase. Sodium azide inhibited the TV-oxide reduction reaction, while aminotriazole did not. With rat liver microsomes, IV-octylamine decreased... 

Figure 4.7. Histamine release from mast cells in response to various dilutions of HRA generated from bovine serum albumin (BSA) by medium derived from stimulated rat neutrophils [156]. Neutrophils ((50-100) x K lml) were stimulated with FMLP (10 5 M), the medium removed and incubated with BSA (10 mg I ml) at pH 4.5 for 18 h at 37° C. It was then boiled and centrifuged (11,000 x g for 30 s), the supernatant fraction was removed, its pH was adjusted to 7.2 and it was added, at various dilutions, to suspensions of mast cells. Histamine release was then measured after 10 min. As the amount of generated HRA was increased histamine release increased to a maximum at 57 4%. Mean + S.E.M., n = 5. Inset Time-course of generation of HRA as assayed by histamine release from isolated mast cells. HRA was generated as before using 50 x 106 neutrophils/ml. Aliquots were removed at the indicated times and assayed (at 50% dilution) for HRA. Note that there is a significant generation of HRA by 2 h. Mean S.E.M., n = 3.

To acquire information on the intrinsic metabolic activity of aquatic organisms, liver of carp (Cyprinus carpio Linnaeus), rainbow trout (Salmo gairdneri) and freshwater snail (Cipango-paludina japonica Martens) was dissected out, homogenized in 0.1M phosphate buffer, pH 7.5, and centrifuged at 105,000 g for 60 min to obtain the microsome-equivalent (described as the microsomal fraction hereafter) fraction. The protein content of microsomal and submicrosomal (supernatant fractions by Lowry s method, microsomal P-450 content ( ), activity of aniline hydroxylase (4) and aminopyrine N-demethylase (5) were determined. 

Figure 1. Top Turbidity, measured at 350 nm, as a function of microtubule polymer mass concentration (expressed as mg/mL polymerized tubulin). Tubulin solutions of varying concentrations were polymerized until they reached stable plateau values in a Cary 118C spectrophotometer. Each sample was then transferred to an ultracentrifuge tube, and microtubules were pelleted, separated from the unpolymerized tubulin in the supernatant fraction, and then resuspended for protein concentration determination. The corresponding turbidity and polymer mass concentrations are plotted here. Bottom Time-course of tubulin polymerization assayed by turbidity.Repro-duced from MacNeal and Purich with permission from the American Society for Biochemistry and Molecular Biology.

A similar activity level was obtained in the deoxycholate, Triton X-100, and NP-40 extract preparations. Octyl glucoside and CHAPS extract preparations showed no detectable prephenate aminotransferase activity. When the hemoglobin step was used, there was no increase in the soluble activity recovered in the initial supernatant fraction, but the specific activity of the deoxycholate (the only detergent tried in this experiment) extract increased about tenfold. We would anticipate equally good results with use of Triton X-100 or NP-40 in combination with the hemoglobin step. 

Microsomal activation for bacterial assays typically has involved crude supernatant fractions (S9) from livers of rats induced by polychlorobiphenyl mixtures, usually Aroclor 1254. These S9 mixtures contain a spectrum of mixed function oxidases and other enzymes active in biotransformation. In such mixtures, a given compound might be activated to become more mutagenic, may be inactivated, or may remain unaffected. All three types of response have been observed with various water residues (9, 14). 

Partially purified preparations of guanine aminohydrolase (EC 3.5.4.3) have been reported from rabbit liver (68,186), rat liver (61), Clostridium addwrici (187), rat brain (60, 188, 189), and lingcod muscle (190). The rat brain enzyme (60) occurs in both the mitochondrial and supernatant fraction the latter fraction yielded two forms, A and B from DEAE-cellulose, which were subsequently purified 70- and 600-fold, respectively. Form B had a specific activity of 290 /unoles/min/mg. Kinetic, immunochemical, and electrophoretic studies revealed that the mitochondrial enzyme was distinct from supernatant enzyme B. A distinction between the supernatant A and B forms was less certain (60). 

In contrast to y-glutamyl transpeptidase, which is almost entirely bound to particulates, y-glutamyl cyclotransferase is present in the supernatant fractions of liver and other tissues. Studies on the distribution of the enzyme shows that it is present in most animal tissues but that there are some species differences (Table IV). Relatively high enzymic activity is found in kidney, liver, brain, and skin. In man, the highest activity is found in the brain. The unusually high activity in... 

Nucleotidase present in 48,000 X Q supernatant fractions of rat and guinea pig skeletal muscle extracts has been examined briefly (7-4). 5 -UMP seems to be the preferred substrate. The enzyme from fish skeletal muscle has also been studied (75). This enzyme hydrolyzes all ribo-and deoxyribonucleoside 5 -phosphates (except dCMP and dTMP) with preference for 5 -IMP and 5 -UMP. The enzyme is strongly activated by Mn2+ Mg2+ is a less powerful activator, and Zn2+ and EDTA are inhibitors. This enzyme thus appears similar to the soluble activity from mammalian liver (88, 86). 5 -Nucleotidase in mammary gland hydrolyzes all 5 -ribonucleotides and shows a decrease from pregnancy to early lactation (76). Rats injected with glucagon show increased 5 -nucleotidase in pancreatic islet tissue (77). The enzyme in mouse kidney has been examined histochemically and electrophoretically and found to exist as isozymes (75). Electrophoretic techniques have also provided evidence that the enzyme exists as isozymes in many other tissues of the mouse such as liver, spleen, intestine, testes, and heart (79). 

It appears certain that there is more than one 5 -nucleotidase present in most mammalian tissues. This is best established for liver. In other cases it has not been possible to determine the exact intracellular origin because of the nonselective extraction procedures used. However, those enzymes isolated from acetone powder preparations of chicken liver and rat liver appear to have properties essentially identical to the enzyme present in 100,000 X ff supernatant fraction of rat liver and therefore may be cytoplasmic in origin. This could also be the case for the intestinal mucosa enzyme. 

Pogell et al. (33) have reported that a natural substance present in liver supernatant fractions, which appears to purify together with phos-phofructokinase activity, also causes an increase in catalytic activity in the neutral pH range. The precise nature of this activator and its possible... 

Fructosediphosphatase has been purified from the supernatant fraction of swine kidney homogenates by a procedure which included chromatography on phosphocellulose and fractionation with ammonium sulfate (20, 65). This preparation was free of other phosphatases, showed high affinity for fructose 6-phosphate, and was inhibited at concentrations of substrate above 10 4 M. The inhibition by higher concentrations of substrate was greater at lower pH this caused the apparent pH optimum to shift from pH 8.5 to 9.5 when the substrate concentration was increased from 5 X 10"5 M to 5 X 10"4 M. The molecular weight was estimated to be approximately 130,000 (65). 

A typical crude broken-cell preparation contains disrupted cell membranes, cellular organelles, and a large number of soluble proteins, all dispersed in an aqueous buffered solution. The membranes and the organelles can usually be separated from one another and from the soluble proteins by differential centrifugation. Differential centrifugation divides a sample into two fractions the pelleted fraction, or sediment, and the supernatant fraction, that is, the fraction that is not sedimented. The two fractions may then be separated by decantation. 

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