Kinetics of gossypol inhibition of bovine lactate dehydrogenase X

Gossypol, a polyphenolic binaphthalene dialdehyde isolated from cotton meal is a potent inhibitor of lactate dehydrogenase-X purified from bovine testis. For the conversion of pyruvate to lactate the IC50 for gossypol is 200 microM for the reverse reaction the IC50 is 12 microM. Gossypol is a competitive inhibitor of NADH, Ki = 30 microM (Km = 17 microM), and NAD+, Ki = 6 microM (Km = 130 microM), and noncompetitive for pyruvate, Ki = 220 microM (Km = 224 microM), and lactate, Ki = 52 microM (Km = 5.6 mM).     

Alpha-foetoprotein and oestrogen metabolism. I. -Influence of alpha-foetoprotein on the metabolism of steroids by rat liver microsomes in vitro.

Rat alpha-foetoprotein (AFP) was shown to inhibit the formation of water soluble metabolities of oestrone and oestradiol by incubation with microsomes from rat liver in the presence of NADPH. The results support the proposal that in young animals the low activity of enzymes responsible of oestradiol metabolism may be due in part to the presence of AFP and not only to the low level of these enzymes.     

Insulin-stimulating peptide from tryptic digest of bovine serum albumin

Insulin-stimulating peptide was isolated from a tryptic digest of bovine serum albumin by gel permeation, SP Sephadex column chromatography, reversed phase HPLC and cation-exchange HPLC. This peptide, with a molecular weight of about 8,400, had no insulin-like activity by itself, but enhanced fatty acid synthesis from glucose in rat adipose tissue explants in the presence of suboptimal concentrations of insulin. It also stimulated the effect of insulin on CO2 production from glucose in rat adipocytes, without affecting insulin binding. These stimulations were dose-dependent and were observed at concentrations of more than 2 X 10(-7) M peptide only in the presence of a suboptimal concentration of insulin.     

1,24,25-Trihydroxyvitamin D3: a circulating metabolite in vitamin D2-treated bovine

Methods have been developed for the examination of yeast RNA polymerases I, II, and III by electron microscopy. The results enabled us to establish the size and shape of a eucaryotic RNA polymerase for the first time. The enzymes are roughly spherical in shape and compact in appearance. Their measured molecular diameters are 12.7 ± 0.4 and 11.0 ± 1.4 (SD) nm for polymerase I, 12.7 ± 1.1 and 12.2 ± 1.0 (SD) nm for polymerase II, and 13.6 ± 0.6 and 11.5 ± 1.3 (SD) nm for polymerase III.     

Properties of partially purified bovine brain dopamine beta-hydroxylase.

Dopamine beta-hydroxylase has been partially purified from bovine brain. A 140-fold purification factor was achieved using solubilization with Triton X-100, ammonium sulphate fractionation between 20-50 per cent saturation, affinity chromatography on concanavalin A-Sepharose 4 B and then filtration through Sephadex G200. The specific activity at the end was 51 nmoles/h/mg protein. The majority of endogenous inhibitors were lost. Immunological studies, kinetic studies, studies on the interaction with lectins and the effect of carboxylic acids on enzyme activity were carried out. Our data are in favour of the close similarity between the bovine brain and adrenal enzymes. No major differences could be found, at least with the characterization experiments using in the present study.     

Hepatic heme metabolism in selenium-deficient rats: effect of phenobarbital.

Hepatic heme metabolism was examined in selenium (Se)-deficient and Se-adequate (control) rats. Administration of phenobarbital stimulated heme synthesis in the liver in both Se-deficient and Se-adequate rats. In contrast to these results, phenobarbital increased microsomal heme oxygenase (MHO) activity six- to eightfold in Se-deficient but not control rats. These data suggest that the previously reported abnormalities of cytochrome P-450 induction in Se-deficient rats are related to increased degradation of hepatic heme.     

The synthesis and deployment of filamin in chicken skeletal muscle

During myogenesis in vitro the actin-binding protein filamin is present in myoblasts and early fused cells and is associated with α-actinin-containing filament bundles, as judged by double immunofluorescence using antibodies specific for these two proteins. Approximately one day after cell fusion, yet before the development of a-actinin-containing Z line striations, filamin disappears from the cells. Later in myogenesis, several days after the appearance of α-actinin-containing Z line striations, filamin reappears and accumulates in the cells. Double immunofluorescence with antibodies to filamin and vimentin (or desmin) reveals that the newly appearing filamin localizes now to the myofibril Z line and is visible there shortly before vimentin or desmin becomes associated with the Z line. Immunofluorescent localization of filamin in isolated chicken skeletal myofibrils and Z disc sheets indicates that filamin has the same distribution as desmin and vimentin; it surrounds each myofibril Z disc and forms honeycomb-like networks within each Z plane of the muscle fiber. Filamin may thus be involved in the transition of desmin and vimentin to the Z disc. Analysis of whole-cell extracts by SDS-polyacrylamide gel electrophoresis and by immunoautoradiography shows that filamin is present in myoblasts and in myotubes early after cell fusion. Concomitant with the absence of filamin fluorescence during the subsequent few days of myogenesis, the quantity of filamin is markedly reduced. During this time, metabolic pulse-labeling with ³⁵S-methionine reveals that the synthetic rate of filamin is also markedly reduced. As filamin fluorescence appears at the Z line, the quantity of filamin and its synthetic rate both increase. The removal of filamin from the cells suggests that filamin either may not be required, or may actually interfere with a necessary process, during the early stages of sarcomere morphogenesis. These results also indicate that the periphery of the Z disc is assembled in at least two distinct steps during myogenesis.     

Structural alterations in fibronectin correlated with morphological changes in smooth muscle cells

Nontransformed cultures of vascular smooth muscle cells proliferate until they form a confluent sheet of cells. Subsequently, the cells become reorganized to form multicellular nodules that are loosely attached to the substrate. The formation of nodules is facilitated by the addition of medium conditioned by nodular cultures. Nodulation is inhibited by the addition of fibronectin. Fibronectins derived from monolayer culture conditioned medium or from plasma are maximally effective while fibronectin isolated from nodular cell conditioned medium is inactive. Analysis by NaDodSO4-polyacrylamide gel electrophoresis reveals that the nodular cell fibronectin has a molecular weight that is about 20-30 kd less than that of monolayer cell fibronectin. Further, nodular cell conditioned medium contains an activity that can convert both plasma fibronectin and monolayer cell fibronectin to the lower molecular weight correlated with the loss of biological activity.     

Ligand-induced alterations in the reactivity of sulfhydryl groups and the structure of bovine liver glutamate dehydrogenase

The fluorogenic probe 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) was employed as an environmentally sensitive reporter label for free sulfhydryl groups of bovine liver glutamate dehydrogenase. A maximum of six -SH groups per subunit was titrated in Tris and borate buffers (pH 7.8) in 2 h but there was no reaction in the presence of phosphate buffer. The rate and extent of -SH reactivity was changed significantly by one of the substrates and some allosteric effectors. Adenosine nucleotides, NADH, and α-ketoglutarate promoted conformational alterations in glutamate dehydrogenase such that -SH groups were rendered virtually unreactive in [enzyme-ADP], [enzyme-NADH], and [enzyme-α-ketoglutarate]binary complexes. GTP, a negative allosteric modulator, showed no effect on -SH exposure. Measurements of protein circular dichroism spectra and catalytic activity in conjunction with -SH reactivity demonstrated a direct relationship between structural stability, biological activity, and ligand-induced conformational changes. The ligands that strongly protected the enzyme from reaction with NBD-C1 concomitantly maintained its structural and functional integrity.     

Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

A spontaneously active (Mr greater than 350,000) and an ATPMg-dependent phosphatase (Mr congruent to 140,000) were identified in bovine aortic smooth muscle. The spontaneously active phosphatase was effective in dephosphorylating both phosphorylase a (240nmol32P/min/mg) and phosphorylated myosin light chains (1000nmol32P/min/mg). In contrast, the ATPMg-dependent phosphatase was only effective in dephosphorylating phosphorylase a (400nmol32P/min/mg). Phosphorylase phosphatase activity of the ATPMg-dependent enzyme was suppressed by the well-characterized modulator protein (inhibitor-2), whereas the activity of the spontaneously active enzyme was unaffected. The aortic spontaneously active phosphatase did not convert to an ATPMg-dependent form when it was stored at 4 degrees or incubated at 30 degrees C in either the presence or absence of modulator protein. These findings suggest that spontaneous and ATPMg-dependent phosphatase activities described in these studies are probably ascribable to different enzymes. Since both phosphorylase and myosin light chains are phosphorylated when smooth muscle contracts these phosphatases may participate in coordinating arterial contractility and metabolism.     

Relative phosphate contents of phosphofructokinase and other soluble phosphoproteins in skeletal muscle

In vivo phosphorylation of muscle proteins has been studied by incorporation of [³²P]phosphate with emphasis placed upon the phosphorylation of glycolytic enzymes. Of the approximately 25 soluble proteins resolved by two-dimensional electrophoresis that contain significant ³²P, phosphofructokinase was the sole glycolytic enzyme identified as a phosphoprotein. The extent of phosphorylation found for this enzyme was the same as determined previously for purified phosphofructokinase and was about the same as the extent of phosphorylation of phosphorylase in resting muscle. Subsequent partial purification of several glycolytic enzymes confirmed the absence of significant amount of phosphate. However, phosphoglycerate mutase contained small amounts of covalently bound ³²P that was exchangeable with 3-phosphoglycerate and therefore, most likely was incorporated during the catalytic reaction cycle. Analogous results were obtained for phosphoglucomutase. Both mutases were also phosphorylated at the same sites by the catalytic subunit of cyclic AMP-dependent protein kinase.     

Altered brain metabolism of testosterone is correlated with reproductive decline in aging quail

In aging quail, an increasing proportion of males show no sexual behavior. A decrease in the mean size of the tests, cloacal gland, and sternotracheal muscles is also observed. In both sexually active and inactive males, plasma testosterone decreases with age but more so in inactive birds. The behavioral and morphological changes observed during aging are correlated with shifts in the intracellular testosterone metabolism resulting in a change in the ratio of active versus inactive metabolites. In the hypothalamus there is a steady decrease with age of 5 beta-reductase activity in all birds and an increase in 5 alpha-reductase activity only in the birds which remain sexually active. In the cloacal gland, the 5 beta-reductase activity markedly increases with age but more so in the birds which become sexually inactive. These data support the notion that the effects of testosterone are controlled by enzymatic shifts which could modulate the sensitivity to the hormone at the cellular level.     

Elimination of insulin-like activity present in certain batches of crude bovine serum albumin by trypsin treatment

A comparative study was carried out in order to determine which of the most commonly used alkalies for protein hydrolysis in tryptophan determination gave the best results. Hydrolyses were performed with 2.5 and 4 n Ba (OH)₂, 4 and 10 n NaOH, 5 n NaOH containing 5% SnCl₂, and with 4 n LiOH, not previously reported for use. The effect of temperature and hydrolysis time on the measured tryptophan content was also determined. Based on results obtained with lysozyme and with seven high protein preparations 4 n LiOH gave the best results. A temperature of 145°C was selected as the most convenient temperature since maximum tryptophan values were obtained with 4–8 h. The hydrolysis time required was inversely related to the protein content of the preparation. Lysozyme, casein, bovine plasma protein, and dehydrated whole egg gave maximum tryptophan content after 4 h hydrolysis while skimmed milk powder, rice flour, wheat flour, and wild legume flour required 8 h hydrolysis.     

Characterization of calmodulin-mediated phosphorylation of cardiac muscle sarcoplasmic reticulum

Sarcoplasmic reticulum, isolated from canine cardiac muscle, was phosphorylated in the presence of exogenous cAMP-dependent protein kinase or calmodulin. This phosphorylation has been shown previously to activate sarcoplasmic reticulum calcium uptake (LePeuch et al. (1979) Biochemistry18, 5150–5157). Calmodulin appeared to activate an endogenous protein kinase present in sarcoplasmic reticulum membranes. The incorporation of phosphate increased with time. However, once all the ATP was consumed, the level of phosphorylated protein started to decrease due to the action of an endogenous protein phosphatase. Dephosphorylation occurred even when the level of phosphorylated sarcoplasmic reticulum remained constant at high ATP concentrations. The phosphorylation of sarcoplasmic reticulum in the presence of calmodulin, increased as the pH was increased from pH 5.5 to 8.5. This phosphorylation was only inhibited by KCl concentrations greater than 100 mm. The apparent K_m of cAMP-dependent protein kinase for ATP was 5.2 ± 0.2 × 10^?5m, and of the calmodulin-dependent protein kinase for ATP was 3.67 ± 0.29 × 10^?5m. Phosphorylation was maximally activated by 5–10 mm MgCl₂; higher MgCl₂ concentrations inhibited this phosphorylation. Thus the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum could be maximally activated at sarcoplasmic concentrations of K⁺, Mg²⁺, and ATP. The calmodulindependent phosphorylation was half-maximally activated at Ca²⁺ concentrations that were significantly greater than those required to promote the formation of the sarcoplasmic reticulum Ca-activated ATPase phosphoprotein intermediate. Thus at sarcoplasmic Ca²⁺ concentrations that might be expected during systole, the sarcoplasmic reticulum calcium pump would be fully activated before any significant calmodul-independent sarcoplasmic reticulum phosphorylation occurred. However, under certain pathological conditions when the sarcoplasmic Ca²⁺ becomes elevated (e.g., in ischemia) the kinase could be activated so that the sarcoplasmic reticulum would be phosphorylated and calcium uptake augmented. Thus, the calmodulin-dependent protein kinase may only function when the heart needs to rescue itself from a possibly fatal calcium overload.     

Patterns of protein synthesis and metabolism during sea urchin embryogenesis

We have analyzed the patterns of protein synthesis in developing embryos of the sea urchin Strongylocentrotus purpuratus by two-dimensional gel electrophoresis. There was an increase in the number of proteins detectably synthesized during development, as well as significant changes in relative rates of synthesis involving approximately 20% of the nearly 900 newly synthesized polypeptides. The majority of these changes were increases rather than decreases in synthesis; about half were of at least 10-fold, while a few were of more than 100-fold. Very few changes were detected upon fertilization and during the first several hours of development, while about 60% of the changes detected occurred between the hatching and the beginning of invagination. An analysis of proteins detected by silver staining indicated that most remained nearly constant in mass during embryonic development, but several increased or declined substantially. Many proteins present in eggs were not detectably synthesized in either eggs or embryos.     

Studies of a reactive histidine of dyphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart.

Diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart was inactivated at neutral pH by bromoacetate and diethyl pyrocarbonate and by photooxidation in the presence of methylene blue or rose bengal. Inactivation by diethyl pyrocarbonate was reversed by hydroxylamine. Loss of activity by photooxidation at pH 7.07 was accompanied by progressive destruction of histidine with time; loss of 83% of the enzyme activity was accompanied by modification of 1.1 histidyl residues per enzyme subunit. The pH-rate profiles of inactivation by photooxidation and by diethyl pyrocarbonate modification showed an inflection point around pH 6.6, in accord with the pK_a for a histidyl residue of a protein. Partial protection against inactivation by photooxidation or diethyl pyrocarbonate was obtained with substrate (manganous isocitrate or magnesium isocitrate) or ADP; the combination of substrate and ADP was more effective than the components singly. As demonstrated by differential enzyme activity assays between pH 6.4 and pH 7.5 with and without 0.67 mm ADP, modification of the reactive histidyl residue of the enzyme caused a preferential loss of the positive modulation of activity by ADP. The latter was particularly apparent when substrate partially protected the enzyme against inactivation by rose bengal-induced photooxidation.     

Effect of a phosphonic acid analog of choline phosphate on phospholipid metabolism in Tetrahymena

When Tetrahymena thermophila is grown on a medium containing increasing concentrations of N,N,N-trimethyl-2-aminoethylphosphonate (TMAEP), up to 60% of the choline phosphate in phosphatidylcholine is replaced by the phosphonic acid. There is an increase in the relative amount of quaternary ammonium-containing lipid (phosphatidylcholine plus TMAEP-lipid) at the expense of phosphatidylethanolamine. There is no effect of the TMAEP on either 2-aminoethylphosphonolipid levels or on de novo 2-aminoethylphosphonate synthesis. Higher levels of TMAEP in the medium (25 and 50 mm) lead to decreased growth of Tetrahymena and to an abnormal cell morphology.     

Effects of pertussis vaccine on the lipid metabolism of hamsters

Administration of pertussis vaccine to hamsters markedly affected their lipid metabolism. Four days after the administration of the vaccine a severe fatty liver was observed. Concomitantly, a rise in the serum levels of free fatty acids, triacylglycerols and ketone bodies was detected. It is suggested that an altered regulation of adipose tissue lipolysis might be at least partially responsible for the observed effects.