Coupling of fructose-1,6-P2 to aminated agarose by Schiff base reduction. Affinity chromatography of yeast aldolase

Summary Fructose-1,6-P₂ was immobilized by sodium borohydride reduction of the Schiff base formed with aminated agarose (AH-Sepharose 4B^®). The coupling occurs with high yield (25 moles immobilized fructose-1,6-P₂ per ml packed gel) at neutral pH and room temperature. Schiff base reduction thus provides a convenient and mild coupling procedure for sugar phosphates preserving their labile phospho ester bonds. As exemplified by a new isolation procedure for fructose-1,6-P₂ aldolase from yeast, sugar phosphates insolubilized in this manner may be used for affinity chromatography of the corresponding enzymes, provided that contaminating unspecific phosphatases are removed in a preceding fractionation step.This work was supported by the Swiss National Science Foundation, grant No. 3.620-0.75. A preliminary account has been presented at the 10th Int. Congress of Biochemistry, Hamburg, 1976, Abstracts. p. 194.Acknowledgment. The excellent technical assistance of Alice Gasser is gratefully acknowledged     

Sul meccanismo della eptoformazione non ossidativa nel muscolo scheletrico

Summary F-1,6-P has been shown to increase the rate of non-oxydative heptoformation from glucose-6-phosphate and fructose-6-phosphate in enzymatic preparations of rat skeletal muscle. The mechanism of this increase — probably due to triose-phosphate, formed by aldolase action on F-1,6-P — is discussed.     

Fructose-1,6-diphosphate reduces acute ECG changes due to doxorubicin in isolated rat heart

Doxorubicin (DXR) (0.17 x 10(-4) M) induces an acute cardiotoxicity in isolated rat heart; there is a progressive widening of the S alpha T segment, with a decrease in force derivatives and in the coronary flow. Concurrent perfusion with fructose-1,6-diphosphate (FDP) (10(-5)-10(-4) M) dose-dependently reduces the S alpha T enlargement but fails to affect the reduction in force derivatives and coronary flow. The target of cardiac protection by FDP might be the ionic mechanisms underlying the action potential configuration.     

Functional consequences of treating turkey liver fructose-1,6-bisphosphatase with penicillin G.

Treatment of turkey liver fructose-1,6--bisphosphatase with penicillin G progressively inactivated the enzyme and desensitized the enzyme toward high substrate inhibition. The treatment also led to reduced sensitivity to AMP inhibition and the loss of cooperative interaction among AMP-binding sites. These altered properties were not reversed by dialysis, but were prevented when treatment with penicillin G was perfomed in the presence of substrate.     

Uptake of glusose-C14 and fructose-C14 by buffalo spermatozoa in extenders during cold storage

Buffalo spermatozoa exhibit insignificant uptake of glucose-C14 and fructose-C14 when semen samples were preserved in cold for 96 h in 2 extenders. Incorporation of C14 in spermatozoa, TCA precipitable proteins and soluble sugar phosphates was either equal or less in semen samples preserved in cold. CAW appeared to be a better extender than SKMEY as revealed by sugar uptake in cold.     

Functional consequences of treating turkey liver fructose-1,6-bisphosphatase with penicillin G

Summary Treatment of turkey liver fructose-1,6-bisphosphatase with penicillin G progressively inactivated the enzyme and desensitized the enzyme toward high substrate inhibition. The treatment also led to reduced sensitivity to AMP inhibition and the loss of cooperative interaction among AMP-binding sites. These altered properties were not reversed by dialysis, but were prevented when treatment with penicillin G was performed in the presence of substrate.This work was in part supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA) and in part by Faculty Research Grant from Southern School of Pharmacy, Mercer University, Atlanta (Georgia, USA).     

Studies on the biochemical effects of glibenclamide on alloxan diabetic rabbits

Treatment of alloxan diabetic rabbits with glibenclamide, the most potent of the sulfonylureas, for a period of 2 months, significantly ameliorated the diabetic condition. It produced a decrease in serum and liver lipids, amino acids, serum urea, blood sugar and urine sugar; increase in body weight, serum and liver proteins, liver glycogen, glucose tolerance and serum and liver acid labile phosphates. The possible mechanism of action of this hypoglycemic agent is discussed.     

Studies on the biochemical effects of glibenclamide on alloxan diabetic rabbits

Summary Treatment of alloxan diabetic rabbits with glibenclamide, the most potent of the sulfonylureas, for a period of 2 months, significantly ameliorated the diabetic condition. It produced a decrease in serum and liver lipids, amino acids, serum urea, blood sugar and urine sugar; increase in body weight, serum and liver proteins, liver glycogen, glucose tolerance and serum and liver acid labile phosphates. The possible mechanism of action of this hypoglycemic agent is discussed.     

Unexpected collagen crosslinking observed during in vitro radiolabeling of the galactosyl moiety

Aldehyde groups, generated by oxidation of free primary alcohol groups of galactosyl residues of glycoproteins by galactose oxidase, can react with free amino groups on the polypeptide chain through Schiff base formation. Subsequent reduction with tritiated borohydride results in the formation of stable crosslinks instead of the expected generation of radiolabeled alcohol groups. Attempts to perform in vitro radiolabeling of collagen type I by this procedure resulted in undesirable crosslinking with profound alteration of the physical properties that rendered the resulting radiolabeled preparation unsuitable for biochemical studies.     

Proinsulin C-peptide and its C-terminal pentapeptide: degradation in human serum and Schiff base formation with subsequent CO<Subscript>2</Subscript> incorporation

Processing of human proinsulin C-peptide and its C-terminal pentapeptide in blood serum was studied using reverse-phase HPLC and electrospray mass spectrometry. The results reveal degradation of both peptides, with a longer half-life for intact C-peptide than for the C-terminal pentapeptide. Products from C-peptide degradation were not distinguishable from the peptide background, suggesting endopeptidase degradation of C-peptide. In contrast, a set of products from the C-terminal pentapeptide were identifiable and corresponded to successive losses from the N terminus, showing that the pentapeptide is degraded by aminopeptidase in serum. Consistent with this finding, a slower degradation was found for the N-acetyl-protected pentapeptide. Removal of serum proteins by acetone precipitation produced N-terminally carbamate-modified C-peptide via a Schiff base intermediate (a ketimine with acetone), to which CO(2) was added and acetone removed, generating a cyclic side chain via anhydride formation. The modification was not seen with the pyroglutamate form of C-peptide, with the N-terminally acetylated C-peptide, or with a control peptide having N-terminal Phe, but was found with human C-peptide, its N-terminal tetrapeptide, and a rat C-peptide fragment (all with N-terminal Glu). Hence, the modification appears to require N-terminal Glu, but this is not the only prerequisite since the C-terminal pentapeptide and another control peptide (also starting with Glu) were not modified. A peptide aldimine Schiff base leading to CO(2) incorporation was detected with formaldehyde in NaHCO(3). The observation that C-peptide forms Schiff bases with ketones/aldehydes, enhancing covalent attachment of CO(2), may have biological implications.     

Uptake of glucose-C14 and fructose-C14 by buffalo spermatozoa in extenders during cold storage

Summary Buffalo spermatozoa exhibit insignificant uptake of glucose-C¹⁴ and fructose-C¹⁴ when semen samples were preserved in cold for 96 h in 2 extenders. Incorporation of C¹⁴ in spermatozoa, TCA precipitable proteins and soluble sugar phosphates was either equal or less in semen samples preserved in cold. CAW appeared to be a better extender than SKMEY as revealed by sugar uptake in cold.Acknowledgment. We are indebted to Dr D. Sundaresan, Director, for his kind encouragement in this study.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K+ or NH+4, increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K+, respectively.     

Novel rearrangement of purines

6-Trichloromethyl-9-methylpurine (1) rearranges to 6-dichloromethyl-9-methyl-8-oxopurine (2) in aqueous mild acidic solution. The rearrangement is rationalized in terms of a reaction involving protonation, covalent hydration, prototropic equilibrium and/or a hydride transfer. An alternative mechanism involving a "positive' halogen compound and hypochlorous acid as an intermediary is also proposed. Compound 1 condenses with 4,5-diaminopyrimidine to give the purine-pyrimidine Schiff base pair 4.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Summary Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K⁺ or NH ₄ ⁺ , increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K⁺, resepctively.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

Effects of long-term feeding of glibenclamide on normal rats

Summary Prolonged administration of glibenclamide decreased blood sugar, liver glycogen and protein and increased liver and serum lipids and organic phosphates of liver in normal rats. A significant weight increase observed in glibenclamide group of rats is attributed to lipid accumulation.     

Decreased monosaccharide transport in renal brush-border membrane vesicles of spontaneously hypertensive rats

Na(+)-dependent D-glucose and D-galactose transport were studied in brush-border membrane vesicles (BBMVs) from kidney cortex isolated from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of Na(+)-dependent D-glucose and D-galactose transport were significantly lower in SHR compared with WKY, the observed decreases being similar for both substrates. To explain the reduction in sugar transport by renal BBMVs, the density of Na(+)-dependent sugar cotransporters was studied in BBMVs from kidney cortex isolated from SHR and WKY rats. Phlorizin-specific binding and Western blot analysis indicated a reduction in the density of the cotransporters in SHR relative to WKY rats. This reduction was similar to those found for the initial rates and accumulation ratios for D-glucose and D-galactose in SHR. Na+ uptake, studied using 22Na+, was significantly increased in SHR, so the observed reduction in sugar transport could be due to disruption of the Na+ gradient between renal BBMVs in SHR. Furthermore, a significant decrease in the activity of Na(+)-K(+)-ATPase was observed in SHR. In conclusion, changes in the density of the Na(+)-dependent sugar cotransporter and in the Na+ gradient across the brush-border membranes might be involved in the observed reduction in sugar transport by renal BBMVs from SHR.     

Beziehungen zwischen Funktion und Stoffwechsel des Zentralnervensystems nach Pervitin

Summary The ATP- and coenzyme-A-content increases in the brain of white mice during excitation produced by Pervitin (2–3µg/g bodyweight) proportional to motility, rate of respiration and oxygen-uptake. These facts, observed in this almost physiological excitation, are contrary to the reduction of the energy-rich phosphates in the brain during convulsions.     

Demonstration of fructose 1,6-bisphosphatase in human term placenta.

A proteolysed form of fructose 1,6-bisphosphatase (Fru-P2ase) has been detected and characterized in human term placenta. The extract was found to contain very low levels of activity with an alkaline pH optimum. Western blotting demonstrated a polypeptide of Mr 26,000, instead of the subunit of Mr 36,000 observed in native mammalian Fru-P2ases.     

Inhibition of liver fructose 1,6-bisphosphatase activity by Zn2+: reversal by imidazole pyruvate.

Imidazole pyruvate was found to be a very potent natural chelating agent in reversing the inhibition of liver fructose 1,6-bisphosphatase activity by Zn2+. This metabolite may play a physiological role in gluconeogenesis.     

On the mechanism of the glycogenolytic effect of dinitrophenol. Activity of phosphofructokinase in perfused hearts.

To explain the mechanism of DNP action the contents of ATP, ADP, AMP and G-6-P were determined in perfused rat hearts in a period of time in which the rate of the glycogenolysis was increased. The levels of these metabolytes in the extract were consistent with an increase of the phosphofructokinase activity. On the other hand, the finding of higher activity of phosphyrylase beta kinase in DNP-poisoned animals could be explained as due to the low content of G-6-P in the perfused hearts subjected to the action of the drug.