bluuebunny/crossref_metadata_2025 huggingface dataset:
Steroids in Germfree and Conventional Rats
Authors: Gustafsson J.‐Å. , Sjövall J.
Published: March 2005

The predominant steroids in faeces from germfree rats were identified by gas chromato‐graphy‐mass spectrometry as 3α,11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one and 3α,15α,21‐trihydroxy‐5α‐pregnane‐11,20‐dione. Smaller amounts of 11β,21‐dihydroxy‐5α‐pregnane‐3,20‐dione, 3α,11β,21‐trihydroxy‐5α‐pregnan‐20‐one, 3α,21‐dihydroxy‐5α‐pregnane‐11,20‐dione (tentatively) and another 15α‐hydroxylated steroid, 3α,15α‐dihydroxy‐5α‐pregnan‐20‐one were also identified. None of the 21‐hydroxylated steroids could be found in faeces from conventional rats. Results are given which indicate that the major 21‐hydroxylated steroids are present as 21‐mono‐sulphates and, to a much smaller extent, as disulphates. It is concluded that 15α‐hydroxylation is a major reaction in the metabolism of steroids in the rat and that the intestinal flora plays an important role in the metabolism of 21‐hydroxylated steroids.
Steroids in Germfree and Conventional Rats
Authors: Gustafsson J.‐Å.
Published: March 2005

Gas chromatography‐mass spectrometry has been used to identify nine C19 and six C21 steroids in faeces from conventional rats. The compounds characterized were: 3β‐hydroxyandrost‐5‐en‐17‐one; 3α,11β‐dihydroxy‐ and 3α,7α‐dihydroxy‐5α‐androstan‐17‐one; 5α‐androstane‐3α,7α,17β‐triol; 5α‐androstane‐3α,15α,17β‐triol; 3α(and 3β),19‐dihydroxy‐5α‐androstan‐17‐one; 5α‐androstane‐3α(and 3β),17β‐diol; 3α(and 3β),16α‐dihydroxy‐5α‐pregnan‐20‐one; 3α(and 3β),15α‐dihydroxy‐5α‐pregnan‐20‐one and 3α(and 3β),19‐dihydroxy‐5α‐pregnan‐20‐one. Their chromatographic behaviour and ready solvolysis in ethyl acetate acidified with sulphuric acid indicate that the steroids were present in faeces as monosulphates. These findings are discussed in relation to the results obtained from analyses of steroids in faeces from germfree rats.
Dissoziation und Rekonstitution der Thiolase
Authors: Gehring U. , Riepertinger C.
Published: March 2005

Thiolase from pig heart shows in dilute salt solutions the following hydrodynamic properties: s°20,w= 7.88 S D°20,w= 4.2 × 10−7 cm2/sec = 0.731 ml/g From these data a molecular weight of 169,000 was calculated which is in good agreement with the value of 168,000 obtained by the Archibald method.

In the presence of 5 M guanidine‐HCl the enzyme dissociates into subunits with a molecular weight in the range of 39,000–44,000 as determined by use of the Archibald method, sedimentation equilibrium analysis and combination of sedimentation velocity with viscosity and diffusion measurements.

The sedimentation coefficient and the molecular weight of the subunits in 5 M urea (s°20,w= 1.86 S, mol. wt. = 43,700) and in 70% formic acid (s°20,w= 1.73 S, mol. wt. = 41,700) are comparable to those obtained in the presence of guanidine‐HCl.

Treatment of the enzyme with a large excess of succinic anhydride caused a splitting into protein particles with a molecular weight of approximately 92,000, corresponding to half the size of the native enzyme.

Quantitative determinations of the NH2‐terminal amino acid residues by reaction with dinitrofluorobenzene and by Edman degradation yielded 4 valine residues per mole of protein.

These results indicate that thiolase from pig heart contains 4 subunits of similar size, each consisting of one polypeptide chain. The subunits are not linked by covalent bonds. Although binding studies using [1‐14C]acetyl‐CoA and iodo‐[1‐14C]acetamide have indicated the presence of at least 3 active sites per mole of enzyme, we propose that the fully active tetramer contains one active site per subunit.

The dissociation of thiolase in 5 M urea is a reversible process. After dilution to a urea concentration of 0.1 M the reconstitution of the tetrameric structure can be followed by enzyme assay and sedimentation analysis. The association of the subunits is dependent on protein concentration, temperature, pH and the nature of the buffer. Under optimal conditions the original enzyme activity can be restored completely.

Thiolase dissociated with 5 M guanidine‐HCl or 70% formic acid could be reactivated in similar experiments to 70% and 50% of the original activity, respectively.

Denaturation by heat or treatment with trichloroacetic acid could also be partially reversed (40%, 70%) after dissolving the precipitated protein in 5 M urea.
5′‐Deoxy‐5′‐Fluoro‐Thymidine, a Biochemical Analogue of Thymidine‐5′‐Monophosphate Selectively Inhibiting DNA Synthesis
Authors: Langen P. , Kowollik G.
Published: March 2005

5′‐Deoxy‐5′‐fluorothymidine (5′‐fluorothymidine) inhibits the DNA synthesis of cells of the Ehrlich ascites carcinoma of the mouse (carcinoma) in vitro, as estimated by the incorporation of radioactive phosphate. About 90 or 50% inhibition is obtained at concentrations of 1 mM or 70 μM, respectively. The synthesis of neither RNA nor protein is inhibited by 5′‐fluorothymidine. The inhibition of DNA synthesis cannot be reversed by thymidine.

In cell‐free extracts obtained from carcinoma cells, 5′‐fluorothymidine strongly inhibits the phosphorylation of thymidine‐5′‐monophosphate, but hardly at all that of thymidine. A large accumulation of thymidine‐5′‐monophosphate is found among the phosphorylation products derived from thymidine in the presence of 5′‐fluorothymidine. Thus, 5′‐fluorothymidine acts as inhibitory analogue of thymidine‐5′‐monophosphate in the thymidine‐5′‐monophosphate kinase reaction. The inhibition of thymidine‐5′‐monophosphate kinase is competitive. The affinity of the analogue for the enzyme is about three times higher than that of the substrate. The enzyme is also inhibited by thymidine, but its inhibitory activity is only one tenth that of 5′‐fluorothymidine. 5′‐fluorothymidine provides protection of the enzyme against inactivation brought about by incubation in the absence of the substrate.

5′‐fluorothymidine is degraded by pyrimidine phosphorylases at a rate of only one sixth that of thymidine.

5′‐fluorothymidine inhibits the multiplication of carcinoma cells in vitro either completely or by 60% at concentrations of 100 or 10 μM, respectively. The increase in fermentation, normally associated with this cell multiplication, is much less influenced by 5′‐fluorothymidine. Thus, the effect of 5′‐fluorothymidine is primarily seen in cell division, but to a less extent in the development of cytoplasmic processes (similar to unbalanced growth).

Since 5′‐fluorothymidine cannot be phosphorylated at C‐5′ it must be assumed to act unchanged as such. This is in contrast to the classical base, or nucleoside, analogues, which, for inhibitory activity, have to be transferred intracellularly into the nucleotides (lethal synthesis). The independence of 5′‐fluorothymidine of lethal synthesis should be of advantage with regard to resistance problems for a possible use of this compound in cancer chemotherapy.
Determination of Specific Radioactivities of Mononucleotides and Glycolytic Intermediates from Mouse Liver after Labelling in vivo with [32P]Orthophosphate
Authors: Till U. , Blume E. , Günther J. , Reich J.G. , Zahn D. , Klinger R. , Jaroszewicz K. , Frunder H.
Published: March 2005

Experimental procedures and conditions are described for the determination of specific radioactivities of 16 different P‐positions in 12 phosphorylated liver compounds after labelling in vivo with [32P]orthophosphate. These data are necessary for calculations of flux rates within the mononucleotides and the glycolytic pathway.

Purification procedures for the following compounds are given: ATP, ADP, GTP, UTP, UDPG, glucose 6‐phosphate, fructose 6‐phosphate, fructose 1,6‐biphosphate, dihydroxyacetone‐phosphate, glycerol 3‐phosphate, phosphoglyceric acid and phosphoenolpyruvate.

The purification of each compound consists of 3–5 chromatographic steps, in most cases including a specific enzymic conversion.

A preparation procedure is described for mouse liver which shows only a negligible artificial isotope exchange between the β‐ and γ‐P‐positions of ATP. This was proved by addition of labelled ATP to non‐labelled livers.

Five and ten min after intravenous injection of carrier‐free [32P]orthophosphate the specific radioactivities of the compounds examined show identical values, thereby excluding compartmentations with isotope kinetic relevance.
Thioredoxin. 6. The Amino Acid Sequence of the Protein from Escherichia coli B
Authors: Holmgren A.
Published: March 2005

Peptide A, the C‐terminal cyanogen bromide fragment of thioredoxin, was degraded with chymotrypsin and pepsin. Partial sequences of 12 chymotryptic and 6 peptic peptides and the N‐terminal tryptic peptide of trifluoro‐acetylated peptide A were determined. The results were used to establish the order of the previously described tryptic peptides of peptide A and lead to the complete amino acid sequence of peptide A.

Previous experiments had established the amino acid sequence of peptide B, the N‐terminal cyanogen bromide fragment of thioredoxin and the present results thus give the complete amino acid sequence of thioredoxin from Escherichia coli B. The molecule contains 108 residues in a single polypeptide chain with a molecular weight of 11,657 as calculated from the sequence. The functional group of the protein occurs in position 32 to 35 and consists of a disulfide bridge formed by two half‐cystine residues separated by a glycine and a proline residue. No metals were found as part of the functional group.
Evidence for an Amino Acid Transport System in Nuclei Isolated from Embryonic Heart
Authors: Klein R. L. , Horton C. R. , Thureson‐Klein Å.
Published: March 2005

Nuclei isolated from embryonic myocardium of the chick possess an ATP phosphohydrolase and are capable of accumulating amino acid in a diffusible form in large excess of environmental levels. The enzyme is of relatively high specific activity and shows preference for ATP compared to other possible substrates. Properties common to both the enzyme and to net amino acid uptake are requirements for ATP, Mg++, Na+ or K+ and a critical level of Ca++. In the presence of Na+ or K+, both l‐ and d‐forms of several amino acids will further stimulate ATP hydrolysis. The latter stimulation by α‐alanine and the net uptake of this amino acid can both be completely inhibited by similar concentrations of ouabain, EGTA and excess Ca++. Ouabain has no effect on basic Mg++‐activated ATP hydrolysis or that stimulated by the addition of Na+ or K+. It is concluded that these nuclei possess an ATP phosphohydrolase‐linked transport system for α‐alanine and probably for other amino acids. Implications of this transport system are discussed relative to the ultrastructure and compartmentalization of the cardiac cell.
The Influence of Inorganic Phosphate, Adenosine Triphosphate and Glucose 6‐Phosphate on the Activity of Liver Glycogen Synthetase
Authors: de Wulf H. , Stalmans W. , Hers H. G.
Published: March 2005

The important activation of liver glycogen synthetase which occurs after a load of glucose or after the administration of glucocorticoids and which could be demonstrated by assaying the enzyme in a concentrated liver homogenate [1,2], is due to the conversion of an inactive or b form of the enzyme into another form called a, which is active under physiological conditions. This conversion is similar to that previously shown to occur in vitro by Segal and his coworkers [7,8]. The native or b form is prevalent in the untreated animal and has a low affinity for UDPG and for glucose 6‐phosphate; it is active only in the presence of glucose 6‐phosphate but this activity is cancelled by Pi, sulfate, sulfite and ATP. Consequently it remains inactive in the concentrated liver homogenate, which is rich in Pi, and also in vivo, due to the intracellular concentrations of Pi and ATP. Glycogen synthetase a has a high affinity for UDPG and for glucose 6‐phosphate; it is stabilized by phosphate, sulfate and sulfite as well as by glucose 6‐phosphate. It is inhibited by ATP but this inhibition is mostly overcome by physiological amounts of Pi. It is therefore fully active in a concentrated homogenate and in vivo. Magnesium ions have on both forms of the enzyme an effect which is similar to that of glucose 6‐phosphate.

Glycogen synthetase a is conveniently measured in a diluted liver homogenate in the presence of 10 mM sulfate; the activities found in these conditions correlate with those measured in a concentrated homogenate and therefore with the rate of liver glycogen synthesis in vivo. in the cell, Pi appears to be the most important activator of the enzyme, whereas glucose 6‐phosphate has only a minor role.
The Role of Glucose, Glucagon and Glucocorticoids in the Regulation of Liver Glycogen Synthesis
Authors: de Wulf H. , Hers H. G.
Published: March 2005

The conversion of liver glycogen synthetase b into a in vivo, induced by glucose or by glucocorticoids, is further enhanced when both treatments are combined. Once activated, the enzyme is rapidly inactivated by an injection of glucagon, epinephrine or cyclic AMP. When given together with glucose, these substances prevent the usual activation of the enzyme.

The sensitivity of the mouse to glucagon was studied by the inactivation of liver glycogen synthetase and by the activation of liver phosphorylase; the first effect was obtained with lower doses than the second. On the other hand, about 100 times more glucagon was required to inactivate the synthetase previously activated by prednisolone than to prevent its activation by glucose. This difference in sensitivity can presumably not be explained by a different rate of destruction of cyclic AMP, as neither the total activity of the diesterase nor its affinity for the cyclic nucleotide is altered by the treatments. No evidence has been obtained suggesting that prednisolone lowers the sensitivity of the synthetase kinase towards cyclic AMP.
Effects of Ethanol Infusion on the Redox State and Metabolite Levels in Rat Liver in vivo
Authors: Rawat A. K.
Published: March 2005

Hepatic levels of several metabolites of the Embden‐Meyerhof pathway, the citric acid cycle and oxidized and reduced substrate pairs of the lactate, α‐glycerophosphate, β‐hydroxybutyrate, malate, and glutamate dehydrogenase systems were measured in freeze clamped livers from fed male rats, before and after ethanol infusion.

Ethanol infusion in fed male rats after 30 min resulted in an increase in the levels of hexosemonophosphates, triosephosphates, fructose 1,6 diphosphate, lactate, α‐glycerophosphate, malate, β‐hydroxybutyrate, and glutamate. [Lactate]/[pyruvate] ratio increased about 3 fold, [α‐glycerophosphate]/[dihydroxyacetonephosphate] 1.5 fold, [malate]/[oxaloacetate], [β‐hydroxybutyrate]/[acetoacetate], and [glutamate]/[2‐oxoglutarate] × [NH4+] about 2 fold.

The levels of pyruvate, acetyl CoA, citrate, and ATP were decreased during ethãnol treatment.

The hepatic levels of glycogen, oxaloacetate, 2‐oxoglutarate, ammonia, aspartate, and acetoacetate remained unchanged, 30 min after ethanol infusion, as compared to values before ethanol infusion.

Hepatic glucose 6‐phosphatase activity was significantly increased after 15 min of ethanol infusion (1.5 g/kg body weight). The glucose 6‐phosphatase activity showed a maximum 15 min after ethanol infusion, the fructose 1,6 diphosphatase activity did not change significantly 15 and 30 min after ethanol infusion. Hepatic acid and alkaline phosphatase activities measured with β‐glycerophosphate as substrate showed no significant changes compared to values from normal rat livers.

The presence of ethanol (8 mM) in the incubation medium showed no significant effect on the [6‐14C]glucose incorporation into glycogen by the liver slices.
Distribution intracellulaire de ľexonucléase acide dans le foie de rat
Authors: van Dyck J. M. , Wattiaux R.
Published: March 2005

Acid exonuclease activity of rat liver has been determined with the help of DNA core as substrate.

The intracellular distribution of the enzyme has been investigated by differential centrifugation of homogenates and compared with those of cytochrome oxidase, acid phosphatase and glucose‐6‐phosphatase used as reference enzymes for mitochondria, lysosomes and endoplasmic reticulum respectively. Acid exonuclease is principally associated with mitochondrial fractions and exhibits a distribution pattern similar to that of acid phosphatase.

After isopycnic centrifugation in a sucrose gradient of a total mitochondrial fraction, acid exonuclease like acid phosphatase shows a relatively flattened distribution curve with a median equilibrium density of about 1.2. The injection to the rat of Triton WR‐1339, which lowers the equilibrium density of lysosomal enzymes has a similar effect on acid exonuclease.

Acid exonuclease exhibits the phenomenom of structure‐linked latency.

It is concluded that acid exonuclease is associated with the lysosomes in rat liver. Separation between acid DNase and acid exonuclease associated with lysosomes can be performed by chromatography on hydroxyapatite of an extract of purified granules. The complementary action of the two enzymes in lysosomal digestion of DNA is illustrated.
The Abnormalities of Lysosomal Enzymes in Mucopolysaccharidoses
Authors: van Hoof F. , Hers H. G.
Published: March 2005

The activity of acid hydrolases on 13 substrates has been measured in the liver of normal human subjects and of 32 patients with various forms of mucopolysaccharidoses. The most important abnormalities may be summarized as follows:

In 3 patients, there was a complete absence of α‐fucosidase and an excess of fucose in the mucopolysaccharide fraction. The enzymatic deficiency extended to the brain, lung, kidney and urine. In the liver there was also a large increase in the activity of several acid hydrolases, mostly of the α‐galactosidase and of the β‐xylosidase.

In 5 patients with pseudo‐Hurler disease (generalized gangliosidosis), the activity of β‐galactosidase, at pH 3.6 was absent and this defect also extended to other tissues. This enzymatic deficiency explains the known accumulation of galactose‐rich mucopolysaccharides and of galactosyl‐N‐acetylgalactosaminyl N‐acetyl‐neuraminyl)‐galactosylceramide (GM1 ganglioside) in the tissues. Several enzymatic activities were greatly elevated, mostly that of α‐fucosidase, α‐galactosidase and N‐acetyl‐β‐glucosaminidase.

In 14 patients with an usual form of Hurler syndrome, the activity of acid β‐galactosidase was markedly reduced and this abnormality extended also to the brain and the derm but not to the kidney, spleen and leucocytes. The N‐acetyl‐β‐hexosaminidases, the β‐glucuronidase and the α‐fucosidase were much more active than normally.

In two siblings only, affected by an unusual type of Hurler syndrome, there was a marked elevation of β‐galactosidase.

The etiological significance of these findings is discussed and a classification of the mucopolysaccharidoses, based on the activity of the lysosomal enzymes in the liver, is proposed.
The Inhibition of the Fatty Acid Synthetase Multienzyme Complex of Yeast by Long‐Chain Acyl Coenzyme A Compounds
Authors: Lust G. , Lynen F.
Published: March 2005

Fatty acid synthetase of yeast was strongly inhibited in vitro by long‐chain acyl coenzyme A thioesters. Increasing the serum albumin concentration in the reaction mixture lowered, but did not prevent, this inhibition. Enzyme kinetic experiments showed that all long‐chain acyl CoA compounds tested were competitive inhibitors of the substrate malonyl CoA. The inhibition with respect to the substrate acetyl CoA and NADPH was non‐competitive. These results support the hypothesis put forth earlier that the end products of the fatty acid synthetase are bound preferentially in the region of the thiol group which is part of the acyl‐carrier‐protein portion of the complex. The data indicate that the long‐chain acyl CoA thioesters may be of importance in the regulation of de novo fatty acid biosynthesis via end product inhibition of the synthetase.
The Puromycin Reaction and its Inhibition by Chloramphenicol
Authors: Cannon M.
Published: March 2005

Puromycin has been used to release nascent peptides from prelabelled Escherichia coli ribosomes. The puromycin reaction on washed ribosomes is partially inhibited by high concentrations of chloramphenicol and may be partially inhibited by low concentrations of chloramphenicol if this is previously bound to ribosomes not engaged in protein synthesis. If crude extracts are allowed to synthesize protein and then chloramphenicol is added during this synthesis ribosomes obtained from such extracts are highly inhibited in the puromycin reaction although only small amounts of chloramphenicol are actually associated with them. The inhibition cannot be reversed by GTP, supernatant fraction, or crude extract and the inhibition is not overcome by lengthy incubation with puromycin. The inhibition may be completely removed in all cases after washing chloramphenicol‐inhibited ribosomes by centrifugation through 15% sucrose.
Metabolic Differentiation of Distinct Muscle Types at the Level of Enzymatic Organization
Authors: Bass A. , Brdiczka D. , Eyer P. , Hofer S. , Pette D.
Published: March 2005

Activity levels of glycogen phosphorylase, hexokinase, triosephosphate dehydrogenase, lactate dehydrogenase, citrate synthase, 3‐hydroxyacyl‐CoA dehydrogenase, glycerolphosphate dehydrogenase (mitochondrial), and hexosediphosphatase have been determined in white (fast) muscles, red (slow) muscles, heart and smooth muscle of higher animals. The activities of these enzymes are taken as relative measures of metabolic capacities. Their ratios are interpreted as representing relations of different metabolic pathways or systems. In all muscles investigated comparable ratios exist for phosphorylase/triosephosphate dehydrogenase (glycogenolysis/glycolysis), glycerolphosphate dehydrogenase/triosephosphate dehydrogenase (mitochondrial glycerolphosphate oxidation/glycolysis), triosephosphate dehydrogenase/lactate dehydrogenase (glycolysis/lactate fermentation), hexokinase/citrate synthase (glucose phosphorylation/citric acid cycle) and 3‐hydroxyacyl‐CoA dehydrogenase/citrate synthase (fatty acid oxidation/citric acid cycle). With respect to the constancy of these ratios, consistent characteristics exist in the organization of the enzyme activity pattern. It is suggested that the more or less invariable coordination of these metabolic systems is not subject to metabolic differentiation. On the contrary, metabolic differentiation is reflected by extreme variations of the following ratios: triose‐phosphate dehydrogenase/3‐hydroxyacyl‐CoA dehydrogenase (glycolysis/fatty acid oxidation), triosephosphate dehydrogenase/citrate synthase (glycolysis/citric acid cycle), lactate dehydrogenase/citrate synthase (lactate fermentation/citric acid cycle), phosphorylase/hexokinase (glycogenolysis/glucose phosphorylation), and hexosediphosphatase/hexokinase (gluconeogenesis/glucose phosphorylation). These variable enzyme activity ratios are discriminative magnitudes and make it possible to discern distinct metabolic types of muscle. White (fast) muscle is characterized by high capacities of glycogenolysis, glycolysis and lactate fermentation, whereas the capacities of glucose phosphorylation, citric acid cycle and fatty acid oxidation are low. Red (slow) muscles, heart and smooth muscle show inverse characteristics. In white (fast) muscle, high activities of hexosediphosphatase and of mitochondrial glycerolphosphate dehydrogenase indicate that gluconeogenesis starting from glycerolphosphate or triosephosphate is probably important in this muscle type, and compensates for its low capacity of glucose phosphorylation.
Ternary Complexes in Solution
Authors: Huber P. R. , Griesser R. , Prijs B. , Sigel H.
Published: March 2005

The stability constants of the ternary Cu2+ complexes, ethylenediamine‐Cu2+‐pyrocatechol and histamine‐Cu2+‐pyrocatechol dianion, were determined. Both complexes are more stable than one would expect on pure statistical reasons. The imidazole‐containing ternary Cu2+ complex is, however, especially stable. For the equilibrium, Cu(histamine)2+ Cu(pyrocatechol)2⇋ 2[(histamine)Cu(pyrocatechnol)], the constant is log X= 4.86 (I= 0.1; T= 25°), while the statistical value is 0.6. These results were compared with those taken (or calculated) from the literature; it is concluded that the π‐system of the ligands is important for such a great increase in stability. Furthermore, the Cu2+‐histamine 1:1 complex selectively binds to O‐ligands rather than to N‐ligands. Thus, the first coordinated ligand has an influence on the kind of the second to be coordinated, i. e. the Cu2+‐histamine 1:1 complex shows discriminating qualities. The significance of these results is discussed with regard to Cu‐proteins, since it is suggested for several enzymes that the imidazole group of histidine is involved with the binding of metal ions.
The Subcellular Distribution of Lysophospholipase in Bovine Adrenal Medulla
Authors: HÖrtnagl Heide , Winkler H. , Hörtnagl H.
Published: March 2005

A study was made of lysophospholipase activity in bovine adrenal medulla. Maximum hydrolysis of lysolecithin was obtained at pH 6.5. The activity of the enzyme was 5.8 μmole × g tissue−1× hour−1.

Differential centrifugation showed that about 90% of the enzyme was confined to particles. 55% of the activity present in the low speed supernatant was recovered in the “large granule fraction”. About 35% sedimented with the microsomes.

Density gradient centrifugation (1.3–2.0 M sucrose) of the large granule fraction revealed that lysosomes and chromaffin granules contained no significant lysophospholipase activity. By the use of a second gradient (1.0–1.6 M), mitochondria could be partly separated from the microsomes which contaminate the large granule fraction. Lysophospholipase appeared to be concentrated in dense microsomal elements.

It is concluded that most, if not all, of the lysophospholipase activity in bovine adrenal medulla is localised in microsomal elements.
On the Interaction of Nucleotides with Basic Polyamino Acids. Cooperative Binding Behavior
Authors: Wagner K. G.
Published: March 2005

The binding of nucleoside monophosphates by poly‐l‐arginine and guanosine monophosphate by poly‐l‐lysine at low ionic strength and pH 7 consists of two phases. At low values of τ (number of bound nucleotides per amino acid residue) the binding behavior can be fitted to the Langmuir isotherm; at high τ values the binding behavior can be fitted to the Langmuir isotherm; at high τ values the binding is cooperative and accompanied by precipitation of a polyamino acid–nucleotide complex. Three different mathematical approaches were used to describe the binding behavior of the cooperative phase. The results indicate that the magnitude of the parameters for nearest neighbor interaction of bound nucleotides parallels the tendency of self‐association of the respective bases. A value of about 2 kcal/mole was determined for the nearest beighbor interaction energy of guanosine 5′ ‐phosphate with poly‐l‐arginine at two different temperatures, and with poly‐l‐lysine. Two approaches yield intrinsic binding constants which, depending on the model chosen, correspond to binding by sites with free neighbor sites or to direct interaction of nucleotides with the polyamino acid. The intrinsic binding constants obtained with poly‐l‐arginine depend upon the nature of the nucleotide base and, as is shown in the case of guanosine 5′ ‐phosphate, upon temperature. The corresponding value for guanosine monophosphate with poly‐l‐lysine is considerably smaller. It is pointed out that interaction of nucleotides with poly‐l‐arginine may be controlled by both the different stacking tendencies of the nucleotide bases and by the direct affinities between bases and polyamino acid.
Sulfhydryl Groups in Human Ceruloplasmin
Authors: Witwicki J. , Zakrzewski K.
Published: March 2005

Sulfhydryl groups in human ceruloplasmin were determined with β‐hydroxyethyl‐2,4‐dinitrophenyl disulfide. In the native ceruloplasmin one sulfhydryl group has been found. In Urea solutions the number of sulfhydryl groups titrated was found to depend on the concentration of urea and on the presence of EDTA. Two sulfhydryl groups were unmasked in 7 M urea alone, and a third after addition of EDTA. The unmasking of sulfhydryl groups in urea was coincident with the bleaching of ceruloplasmin and with the loss of its oxidase activity.
Sterods in Newborns and Infants
Authors: Gustafsson J.‐Å. , Shackleton C. H. L. , Sjövall J.
Published: March 2005

The steroids in faeces from infants less than one year of age have been studied. Two fractions, monosulphates and “diconjugates”, were separated by chromatography on Sephadex LH‐20. The steroids were liberated by solvolysis and were studied by thin‐layer chromatography and gas‐liquid chromatography‐mass spectrometry. Eighteen steroids, most of them having a 3β,16‐dihydroxy‐Δ5 structure, were identified in the monosulphate fraction of pooled faeces from infants 1–4 months of age. Of these, androst‐5‐ene‐3β,16β,17α(and)17β‐triols, 3β‐hydroxypregna‐5,16‐dien‐20‐one, 3β,16β‐dihydroxypregn‐5‐en‐20‐one, 3α,16α‐dihydroxy‐5β‐pregnan‐20‐one, pregn‐5‐ene‐3β,16α,20α‐triol, pregn‐5‐ene‐3β,20α,21‐triol and pregn‐5‐ene‐3β,16α,20α,21‐tetrol have not been reported as occurring in urine from infants. In addition, eight steroids were found, the stereochemistry of which was not confirmed. Four of the 16‐hydroxylated steroids were also present in the “Diconjugate” fraction.

In contrast to faeces from the younger infants, pooled faeces from infants 6–12 months of age contained only small amounts of steroids, mainly pregn‐5‐ene‐3β,20α‐diol and pregn‐5‐ene‐3β,17α,20α‐triol. The results indicate that significant amounts of steroids are excreted in faeces during the first few months of life, and that metabolism of these steroids by the intestinal flora is of minor importance in this period.
Regulation of the Level of Key Enzymes of Glycolysis and Gluconeogenesis in Liver
Authors: Sillero A. , Sillero M. A. G. , Sols A.
Published: March 2005

Diets rich in fructose, glycerol, or both were fed to normal and diabetic rats, and the activities of enzymes which catalyze irreversible steps of glycolysis and gluconeogenesis were studied in liver. With these diets, rich in precursors of triosephosphates, the activities of glucose‐6‐phosphatase and pyruvate kinase in liver were high, while those of glucokinase and phosphoenolpyruvate carboxykinase were low. A metabolic bifurcation to glycolysis and gluconeogenesis, below and above the triosephosphates level respectively, has then been shown to be easily maintained in liver.

It is suggested that the induction of glucokinase and pyruvate kinase in diabetic animals after insulin administration is a sequential process that involves hormonal induction of glucokinase by insulin and secondary metabolite induction of the L isoenzyme of pyruvate kinase by some glycolytic intermediate.

The occurrence of independent mechanisms for the regulation in liver of the activity of enzymes which catalyze irreversible steps of glycolysis and gluconeogenesis affords a valuable metabolic plasticity.
The Proportion of the Incorporation of Leucine into Albumin to that into Total Protein in Rat Liver and Hepatoma Morris 5123 TC*
Authors: Schreiber G. , Rotermund H.‐M. , Maeno H. , Weigand K. , Lesch R.
Published: March 2005

Labelled serum protein appeared in the blood of normal and Morris hepatoma 5123 TC bearing rats 14 to 15 min after intracaval injection of radioactive leucine. Within this time albumin synthesis can be studied separately in vivo in liver and hepatomas.

Tumor bearing rats were killed within 10 to 14 min after injection of [3H] or [14C]leucine. Postmitochondrial supernatants were prepared from 14C‐labelled liver and 3H‐labelled tumor, treated with sodium deoxycholate and combined. Albumin was purified from the mixture. The ratio [3H]protein to [14C]protein decreased during purification from 7 to 1.2, suggesting that the tumor synthesized less albumin compared to total protein than the liver.

Radiochemically pure albumin was isolated from liver and tumor homogenates from rats killed within 12 to 14 min after injection of l‐[1‐14C]leucine. The specific radioactivity in albumin isolated from liver was 13 times higher than in that from tumor.

The proportion of the incorporation of l‐[1‐14C]leucine into albumin to that into total protein was found to be 3.3% for liver and 0.8% for hepatoma homogenates.
Hemoglobin‐LeporeBaltimore, a Third Type of a δβ Crossover (δ50, β86)
Authors: Ostertag W. , Smith E. W.
Published: March 2005

A new Lepore‐type hemoglobin in a person of Negro descent has been identified. In this person a minor hemoglobin (Hb‐LeporeBaltimore) was detected. An abnormal δβ‐chain was isolated and fingerprinted. This hybrid‐type protein presumably arose as a consequence of unequal crossing over between the δ and β genes, as was concluded for Hb‐LeporeBoston and Hb‐LeporeHollandia. In the new Lepore‐type hemoglobin described here the crossover could be placed between residues δ50 and β86. This type of crossover should occur with the highest frequency if crossing over is equally frequent between any nucleotide and if selection favours none of the crossover types.
Die Synthese verschiedener Carbonsäuren durch den Multienzymkomplex der Fettsäuresynthese aus Hefe und die Erklärung ihrer Bildung
Authors: Sumper M. , Riepertinger C. , Lynen F. , Oesterhelt D.
Published: March 2005

The purified multienzyme complex, fatty acid synthetase of yeast, produces palmityl‐CoA and stearyl‐CoA in equal amounts under standard conditions, with adequate NADPH, and a 1:2 ratio of acetyl‐CoA and malonyl‐CoA. Based on the known enzymatic properties of fatty acid synthetase a model which rationalizes the chain termination at the level of C16 and C18‐acid is proposed.

The model is based on two assumptions that are supported by experimental evidence: (a) the probability of any covalently enzyme‐bound saturated acyl residue forming a product by transfer to CoA is determined by the relative velocities of the condensing and transferring reactions. (b) The growing alkan chain interacts with the enzyme protein only after a chain length of 13 carbon atoms has been attained; this interaction changes the relative velocities in favour of product formation by an energy increment of ‐0.9 kcal for each additional methylene group beyond the 13th.

To calculate the probability of product release at a particular chain length, an equation was derived from the model describing quantitatively the observed product distribution.

The formula suggests conditions under which either short acyl‐CoA derivatives or exclusively stearyl‐CoA can be produced. Synthesis under these conditions was examined experimentally and results indicated that the formula can be applied to a wide range of experimental conditions.

Because the condensation reaction in fatty acid synthesis is the rate limiting step under normal conditions, saturated acyl residues have the longest life‐time as intermediates and consequently the highest probability of leaving the enzyme complex as an endproduct. The reduction reactions can be made rate limiting when the NADPH level drops. Under such circumstances β‐ketoacyl‐CoA's and α,β‐unsaturated acyl‐CoA's appear as endproducts. With NADH as electron donor the first reduction is slowed to such an extent that predominantly β‐ketoacyl‐CoA's derivatives are synthesized.

The possibility that the enzyme complex catalyses synthesis of short chain fatty acids and and β‐ketoacids in vivo is discussed.
Initiation of Hemoglobin Synthesis with Rabbit and E. coli tRNA in a Reticulocyte Cell‐Free System
Authors: Gonano F. , Baglioni C.
Published: March 2005

The mechanism of chain initiation has been investigated in experiments with intact reticulocytes and with a cell‐free system prepared from these cells. Polyribosomes were isolated from reticulocytes incubated with labeled amino acids. The NH2‐terminal amino acid of polyribosome‐bound peptide chains has been analyzed by different methods. No evidence has been obtained for the presence of a blocked NH2‐terminal in polyribosome‐bound peptide chains. Moreover, only valine could be detected at the NH2‐terminal. Globin chains were labeled in cell‐free synthesis with [14C]valyl‐tRNA prepared from rabbit liver or E. coli. Valine was incorporated into the NH2‐terminal as well as into internal positions with both tRNAs. This indicated that a valine codon corresponds to the NH2‐terminal amino acids of rabbit globin since this codon can be recognized by E. coli tRNA. Acetyl‐valyl‐tRNA was not utilized by the cell‐free system nor had it any stimulatory activity on the incorporation of valine. These results are discussed in view of the observation that acetyl‐valyl‐tRNA seems to be required for the translation of hemoglobin messenger RNA in a cell‐free system prepared from E. coli.
Formation and Structure of Gels and Fibrils from Glucagon
Authors: Beaven G. H. , Gratzer W. B. , Davies H. G.
Published: March 2005

Glucagon in acid solution aggregates to produce gels and ultimately fibrils of β‐chains. The kinetics of the aggregation have been followed by viscometry, and are found to exhibit a sigmoidal profile with a long lag phase, associated with the formation of nuclei for polymerisation. This has been demonstrated by the elimination of the lag when fresh solutions are seeded with a preformed glucagon gel. The aggregation is promoted by salt, increased pH (within the acid range of solubility) and increased temperature up to 30°. The addition of 5% of dioxan completely inhibits the aggregation. The interaction has evidently a large hydrophobic driving force, and is opposed by coulombic repulsion. The latter effect can be minimised by acetylation of the amino groups, when the polymerisation is greatly accelerated. This also occurs when only the terminal amino group is blocked by carbamylation. By contrast esterification of the carboxyl groups completely prevents aggregation. At the working pH this does not involve a change in charge, and thus appears to be a specific steric effect. Optical rotatory dispersion shows no conformational difference between native glucagon and the derivatives. The transition from a gel to a fibrillar form is promoted by salt and increased temperature. A possible explanation for the appearance of the fibrillar form in the electron microscope is that it contains tubular structures 50–70 Å in diameter, with about 40 extended chains around the circumference lying in antiparallel manner along the axis.
Short‐Term Effects of Calcium, Potassium, and of Ouabain on Metabolite Levels in the Frog Heart in vivo
Authors: Arese P. , Bosia A. , Rossini L.
Published: March 2005

The short‐term effects of calcium (7.2 mM), potassium (10.7 mM), and of ouabain (0.1%) have been studied in the frog heart in vivo. No dynamic alterations occurred at 30 sec, whereas positive inotropism was observed at 60 sec in the high‐Ca and ouabain treatment; in all cases heart arrest occurred at 120 sec. 18 metabolites (glucose, glucose‐1‐P, glucose‐6‐P, fructose‐6‐P, fructose‐1,6‐P2, dihydroxyacetone‐P, glyceraldehyde‐3‐P, α‐P‐glycerol, pyruvate, lactate, oxaloacetate, malate, α‐ketoglutarate, P‐creatine, creatine, ATP, ADP, AMP) and 17 added parameters (mass‐action ratios of the phosphoglucomutase, phosphohexose isomerase, triosephosphate isomerase, phosphofructokinase, aldolase, adenylate kinase, creatine kinase; redox ratios; ratios P‐creatine/creatine, ATP/ADP; sums P‐creatine + creatine, ATP + ADP + AMP, lactate + pyruvate) were computerized by the stepwise discriminant analysis. Highly significant discrimination between control and treated hearts (thanks to the sensitivity of the statistical method employed) shows that ionic shifts and ouabain elicited modifications of the metabolite pattern which always preceded the effects on heart contractility.

The most meaningful results were as follows:

High calcium effect: At 30 sec, the phosphofructokinase‐catalyzed step and the pyruvate, α‐P‐glycerol and oxaloacetate‐depleting reactions are activated; the P‐creatine and ATP balance is negative; ADP and AMP are increased. At 60 and 120 sec the phosphorylase system, phosphofructokinase, and the whole glycolytic sequence are activated.

High‐potassium effect: At 30 sec the phosphofructokinase‐step and the glycolytic flux are activated. Glucose entry into the cell is impaired. P‐creatine, ATP, AMP, ATP/ADP, P‐creatine/creatine are increased. At 60 sec, all glycolytic substrates are increased.

Ouabain effect: At 30 sec glucose extraction from the perfusing fluid decreases, lactate, pyruvate, α‐P‐glycerol and malate accumulate; P‐creatine and ATP are increased. At 60 sec there is no evidence for activation of the phosphorylase system; the glycolytic pattern points to activation of glucose extraction and of the glucose shunt, and to inhibition of the glyceraldehyde‐3‐P dehydrogenase and 3‐P‐glycerate kinase steps. P‐creatine, ATP, and AMP are decreased, creatine and ADP increased. The partial overlapping of the high‐calcium and ouabain patterns suggests that the glycoside not only raises the intracellular free‐calcium level but also depresses the glyceraldehyde‐3‐P dehydrogenase, 3‐P‐glacerate kinase and adenylate cyclase activity. The observed effects are only partially in accord with the inhibition of Na+‐K+‐dependent adenosinetriphosphatase.
Stimulation of Phospholipid Synthesis in Isolated Rat Liver Mitochondria after Treatment in vivo with Tri‐Iodothyronine
Authors: Kaiser W. , Bygrave F. L.
Published: March 2005

The incorporation in vitro of labelled choline into mitochondria isolated from the livers of normal, hypothyroidic and hyperthyroidic rats was studied. Incorporation into the mitochondria from livers of hyperthyroidic rats was stimulated 2‐ to 3‐fold while that into mitochondria isolated from livers of hypothyroidic rats was depressed about 2‐fold.

Stimulation of choline incorporation into isolated rat liver mitochondria by administration in vivo of triiodothyronine was shown to parallel that of amino acid incorporation.

The direct addition of tri‐iodothyronine to the incubation medium did not stimulate but inhibited the incorporation in vitro of choline into mitochondria isolated from the livers of normal rats.

The data provide further evidence that the measurements of phospholipid precursor incorporation into isolated mitochondria carried out in this and in previous studies, reflect the biosynthetic activity of the organism as a whole.
Hydrolysis of Vitamin A Acetate by Unspecific Carboxylesterases from Liver and Kidney
Authors: Bertram J. , Krisch K.
Published: March 2005

Vitamin A acetate is hydrolysed by highly purified carboxylesterases (EC 3.1.1.1) from liver and kidney of several species (pig, ox, man), whereas long‐chain fatty acid esters of vitamin A are not split.

The hydrolysis of vitamin A acetate by pig liver esterase was characterized further by means of pH optimum, Km, specific and molecular activities and the behavior towards several inhibitors.
Fatty Acid Synthesis from Glucose and Acetate and the Control of Lipogenesis in Adipose Tissue
Authors: Del Boca J. , Flatt J. P.
Published: March 2005

Epididymal fat pads of fed and 48 h fasted rats were incubated in vitro with 10 mM glucose and 0, 60, 120, or 104μunits of insulin/ml. A comparison was made of the metabolism of glucose in the absence or presence of 15 mM acetate in the incubation medium.

At physiological concentrations of insulin, the addition of acetate increases glucose uptake, glucose oxidation to CO2, and incorporation of glucose‐C into fatty acids; concomitantly the rates of glycerol release and lactate production are decreased, and the lactate/pyruvate ratios are lowered. Fatty acid synthesis in the presence of glucose plus acetate was in all cases significantly higher than with glucose alone.

It is concluded that, at a given insulin concentration, the conversion of carbohydrate to fat in intact adipose tissue is limited by the tissue's ability to supply acetyl‐CoA for fatty acid synthesis, and not by the enzymes directly engaged in the synthesis of fatty acid from acetyl‐CoA. The effects of acetate were of greater importance in adipose tissue from rats fasted for 48 h than from fed rats. The impairment in lipogenesis caused by fasting appears to be due primarily to a decrease in the tissue's ability to produce acetyl‐CoA from glucose.
Cyanogen Bromide Peptides from the αl‐Chain of Cod Skin Collagen
Authors: Laszlo F. , Olsen B. R.
Published: March 2005

The α1‐chain of cod skin collagen has been cleaved with cyanogen bromide.

The resulting peptides have been isolated and purified by chromatography on phosphocellulose and CM‐cellulose and gel filtration on Sephadex.

The amino acid composition and molecular weights of eight of these peptides, accounting for about 70% of the whole α1‐chain, are presented.

Comparison of amino acid compositions suggests a homology between three of these peptides and cyanogen bromide peptides from rat skin and chick bone α1‐chains.
The Influence of Ribosomal Factors during the Maturation of Reticulocytes
Authors: Herzberg M. , Danon D. , Revel M.
Published: March 2005

During maturation of reticulocytes their ribosomes become less active for protein synthesis. Testing the stimulation effect of KCl ribosomal wash (KCl factors) extracted from ribosomes from cells of different ages isolated by “density distribution of cells” technique, we show that active KCl factors are present on ribosomes from young cells and not on ribosomes from old cells. We demonstrate that the KCl factors can form polysomes from monosomes. The level of action of these factors is investigated and the possibility that they act as initiation factors is examined.
Effects of Bromopyruvate on the Control and Catalytic Properties of Glutamate Dehydrogenase
Authors: Baker J. P. , Rabin B. R.
Published: March 2005

When assayed with NAD, the time course of inactivation of glutamate dehydrogenase by bromopyruvate is biphasic. This analyses as two simultaneous first order reactions: the faster corresponds to destruction of an NAD activating site and the slower to the destruction of the active site. Both reactions are first order in bromopyruvate and both sites are protected by combinations of coenzymes and regulators.

When assayed with NADP, activity loss is essentially a single first order process. When ADP is included in this assay, the activity loss profile is similar to that observed with NAD. With GTP included in this assay, the activity first increases, corresponding to a partial loss of GTP response, and then falls off in a first order reaction. The loss of response to both regulators is analysed in terms of a model in which approximately 50% of the regulatory sites are lost in a first order reaction.

Evidence is presented that the sites binding ADP and GTP are not identical.
20,25‐Diazadehydrocholesterolester aus den Vesikeln des sarkoplasmatischen Reticulum von Ratten nach peroraler Gabe von 20,25‐Diazacholesterol
Authors: Seiler D. , Kuhn E.
Published: March 2005

In rats with myotonia induced by peróral application of 20,25‐diazacholesterol this substance is enriched as esters of a dehydrogenated product in the vesicles of sarcoplasmic reticulum. The isolation and purification of this product is described. Gas chromatographic analysis of the fatty acid components of this product shows that it is rich in unsaturated fatty acids especially in arachidonic acid. The double bond of the dehydrogenated steroid is located in the sidechain of the originally 20,25‐diazacholesterol as is shown by mass spectrometry. This alteration of the vesicles of sarcoplasmic reticulum may be related to the observed myotonia.
A Comparative Study of Histone Fractions FS(a)1 and F2(a)2
Authors: Leclerc J. , Martinage A. , Moschetto Y. , Biserte G.
Published: March 2005

We have studied the characteristics of the histone fractions F2(a)1 and F2(a)2 from eight different tissues including tumours. We were unable to detect any significant differences either in their behaviour in polycarylamide and starch gel electrophoresis or in their amino acid compositions.