Putrescine-oxidase activity in adult bovine serum and fetal bovine serum.

Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a K_M for putrescine of 2.58×10⁻⁶ m and a V_max of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a K_I of 1.8×10⁻⁸ m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The V_max for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the K_M for putrescine, 50.3×10⁻⁶ m. The K₁ of the ABS putrescine oxidase for aminoguanidine was 41×10⁻⁶ m. On the basis of both the K_M and K_I values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.     

Polyamine cytotoxicity in the presence of bovine serum amine oxidase

The toxicity of extracellular spermine, determined in the presence of fetal calf serum, was studied using three cell lines: FM3A, L1210, and NIH3T3 cells. Amine oxidase in fetal calf serum produces aminodialdehyde generating acrolein spontaneously, H(2)O(2), and ammonia from spermine. Spermine toxicity was prevented by aldehyde dehydrogenase, but not by catalase. Similar concentrations of spermine and acrolein were needed to produce toxicity. Other aldehydes (formaldehyde, acetaldehyde, and propionaldehyde) and hydrogen peroxide were less toxic than acrolein. Spermidine and 3-aminopropanal, which produces acrolein, also exhibited severe cytotoxicity. The degree of cytotoxicity of spermine, spermidine, and 3-aminopropanal was nearly parallel with the amount of acrolein produced from each compound. Thus, it was deduced that acrolein is a major toxic compound produced from polyamines (spermine and spermidine) by amine oxidase.     

Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches

The recently emerging atypical bovine pestiviruses have been detected in commercial foetal bovine serum (FBS) of mainly South American origin so far. It is unclear how widely the viruses are presented in commercial FBS of different geographic origins. To further investigate the possible pestivirus contamination of commercially available FBS batches, 33 batches of FBS were obtained from ten suppliers and analysed in this study for the presence of both the recognised and the atypical bovine pestiviruses. All 33 batches of FBS were positive by real-time RT-PCR assays for at least one species of bovine pestiviruses. According to the certificate of analysis that the suppliers claimed for each batch of FBS, BVDV-1 was detected in all 11 countries and BVDV-2 was detected exclusively in the America Continent. The atypical pestiviruses were detected in 13 batches claimed to originate from five countries. Analysis of partial 5'UTR sequences showed a high similarity among these atypical bovine pestiviruses. This study has demonstrated, for the first time that commercial FBS batches of different geographic origins are contaminated not only with the recognised species BVDV-1 and BVDV-2, but also with the emerging atypical bovine pestiviruses.     

Receptor-mediated insulin degradation and insulin-stimulated glycogenesis in cultured foetal hepatocytes.

Insulin-stimulated glycogenesis and insulin degradation were studied simultaneously at 37 degrees C in cultured foetal hepatocytes grown for 2-3 days in the presence of cortisol. Degradation of cell-associated insulin, as measured by trichloroacetic acid precipitation, was significant after 4 min in the presence of 1-3 nM-125I-labelled insulin. This process became maximal (30% of insulin degraded) after 20 min, a time when binding-state conditions were achieved. No insulin-degradative activity was detected in a medium that had been exposed to cells. At steady-state, the appearance of insulin degradation products in the medium was linearly dependent on time (1.5 fmol/min per 10(6) cells at 1nM-125I-labelled insulin). Chloroquine (3-50 microM), bacitracin (0.1-10 mM) and NH4Cl (1-10 mM) inhibited insulin degradation as soon as this became detectable and caused an increase in the association of insulin to hepatocytes after 20 min. Lidocaine and dansylcadaverine had similar effects, whereas N-ethylmaleimide, aprotinin, phenylmethanesulphonyl fluoride and leupeptin were found to be ineffective. Chloroquine, and also bacitracin, at concentrations that inhibited insulin degradation, decreased the insulin-stimulated incorporation of [14C]glucose into glycogen over 2 h. This effect of chloroquine was specific, since it did not modify the basal glycogenesis, or the glycogenic effect of a glucose load in the absence of insulin. It therefore appears that the receptor-mediated insulin degradation (or some associated pathway) is functionally related to the glycogenic effect of insulin in foetal hepatocytes.     

Polyamine uptake by bovine adrenocortical cells

Bovine adrenocortical cells of fasciculo-reticulata origin in primary culture actively accumulate polyamines from the extracellular medium in an energy-dependent process. At low extracellular concentration (e.g., 1 microM putrescine), the transport system resulted in a several-hundred-fold concentration of polyamine in the cellular compartment within 1-2 h of incubation. Putrescine uptake appeared to be the sum of a sodium-dependent, saturable process, with an apparent Km of about 10 microM and of a non-saturable, sodium-independent component. By contrast, spermine was taken up by the cells mostly in a sodium-independent manner. Cross-competition experiments suggested that both polyamines were at least partly transported by the same system. Using specific corresponding probes, it was shown that the polyamine uptake was independent of the amino acid transport systems of the A, L and N types known in a number of cell systems. Adrenocortical cell polyamine content is known to be modulated by adrenocorticotropin through induction of ornithine decarboxylase activity. The existence of a specific uptake system in these cells opens the possibility of a more rapid pathway for the regulation of cellular polyamine levels. It remains to be examined whether this polyamine transport system is under hormonal control, and whether this can support the suggestion that polyamines may represent a form of intracellular messengers in the mechanism of hormone action.     

Isolation and cultivation of Plasmodium falciparum using adult bovine serum

RPMI 1640 medium supplemented with adult bovine serum and hypoxanthine was superior to human serum-supplemented medium for the isolation of new strains of Plasmodium falciparum in Sudan. Similar observations in Indonesia have since confirmed our results. The chloroquine sensitivity of new isolates was identical in either human or bovine serum. Once acclimated to culture conditions P. falciparum strains grew better when using human serum. Erythrocyte-specific antibody present in adult bovine serum slightly inhibited merozoite invasion of uninfected cells. Removal of this cross-reactive antibody from bovine serum increased parasite multiplication to the level obtained in human serum.     

The suppression of endogenous protein degradation by fractions of foetal calf serum: dialysed serum is less able to suppress degradation in aged cells

We have studied the effect of various fractions of foetal bovine serum upon the endogenous degradation of long labelled proteins in cultured MRC5 cells, and upon other cellular functions. Only heat-inactivated serum was capable of suppressing protein degradation to a similar extent to complete serum. Acid-treated and delipidized sera were moderately effective. Albumin on its own was able to replace 40 per cent of the effect of serum, indicating the exogenous protein might compete with endogenous protein for degradation in lysosomes. Albumin was not capable of supporting DNA synthesis. Dialysed serum showed an age-related effect suppressing protein degradation to a lesser extent and being less effective in supporting DNA synthesis or cellular proliferation in aged cells. All the effects noted were related to lysosomal protein degradation. Serum diffusate did not suppress protein degradation.     

Enzymatic degradation of and bovine serum albumin release from starch-acetate films

The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.     

Glycoprotein expression in foetal and adult mouse cerebellum

1. Explants of cerebellum from foetal mouse were cultured in vitro for up to 12 days. Some glycoprotein components displayed time-dependent changes in concentration in the cultured explants. 2. The specific activity of several enzymes involved in the biosynthesis or degradation of N-linked glycoproteins, increased markedly in the cerebellar explants as a function of time in culture. 3. Glycoprotein expression in foetal mouse cerebellum is compared with that in the adult tissue.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

Polyamine levels in normal human serum. Comparison of analytical methods

A comparison of levels of the polyamines, putrescine, spermidine and spermine, in individual and pooled normal human serum, as determined by three independent methods, high pressure cation-exchange chromatography (HPCC), radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS), has been made. Generally good correlations were found for values derived by the three different methods.     

Polyamine synthesis in mammalian tissues. Isolation and characterization of spermidine synthase from bovine brain.

Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5')-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5'-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.     

Differences in degradation processes for insulin and its receptor in cultured foetal hepatocytes.

Binding and degradation of 125I-labelled insulin were studied in cultured foetal hepatocytes after exposure to the protein-synthesis inhibitors tunicamycin and cycloheximide. Tunicamycin (1 microgram/ml) induced a steady decrease of insulin binding, which was decreased by 50% after 13 h. As the total number of binding sites per hepatocyte was 20000, the rate of the receptor degradation could not exceed 13 sites/min per hepatocyte. Cycloheximide (2.8 micrograms/ml) increased insulin binding by 30% within 6 h, an effect that persisted for up to 25 h. This drug had a specific inhibitory effect on the degradation of proteins prelabelled for 10 h with [14C]glucosamine, without affecting the degradation of total proteins. Chronic exposure to 10 nM-insulin neither decreased insulin binding nor modified the effect of the drugs. The absence of down-regulation of insulin receptors cannot be attributed to rapid receptor biosynthesis in foetal hepatocytes. Cellular insulin degradation, which is exclusively receptor-mediated, was determined by two different parameters. First, the rate of release of degraded insulin into the medium was 600 molecules/min per hepatocyte with 1 nM labelled hormone, and increased (preincubation with cycloheximide) or decreased (tunicamycin) as a function of the amount of cell-bound insulin. Secondly, the percentage of cell-bound insulin degraded was not changed by the presence of protein-synthesis inhibitors (25-30%). The stability of insulin degradation suggested that this process was dependent on long-life proteinase systems. Such differences in degradation rates and cycloheximide sensitivity imply that hormone- and receptor-degradation processes utilize distinct pathways.