bluuebunny/crossref_metadata_2025 huggingface dataset:
The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme)
Authors: Hooper N M , Kenny A J , Turner A J
Published: August 2015

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.
Isolation and characterization of six human hepatic isometallothioneins
Authors: Hunziker P E , Kägi J H R
Published: August 2015

Human hepatic metallothionein (MT) was separated into six isoforms by using reversed-phase h.p.l.c. at the analytical and preparative levels. By comparison with the h.p.l.c. elution profiles of the charge-separable species MT-1 and MT-2 isolated by the procedure of Bühler & Kägi [(1974) FEBS Lett. 39, 229-234], five of these isoproteins are identified as hitherto unresolved subforms of MT-1, and one is identical with MT-2. The six isoforms have distinct and reproducible retention times at neutral pH, where the metal remains bound to the protein, and at low pH, where the metal is removed. Their amino acid compositions display the high cysteine content and the lack of aromatic amino acids and of histidine typical of mammalian metallothioneins, but they differ significantly with respect to all other amino acids. A survey of autopsy material indicates that in adult human liver all six isoforms are usually expressed, albeit in somewhat variable relative proportions.
Genomic organization of human lactate dehydrogenase-A gene
Authors: Chung F Z , Tsujibo H , Bhattacharyya U , Sharief F S , Li S S-L
Published: August 2015

A human genomic clone containing the lactate dehydrogenase-A (LDH-A) gene of approx. 12 kilobases in length was isolated and characterized. The protein-coding sequence is interrupted by six introns, and the positions of these introns are at the random coil regions or near the ends of secondary structures located on the surface of the LDH-A molecule. An additional intron is present at 24 nucleotides 5′ to the translation initiation codon ATG, while the 3′ untranslated sequence of 565 nucleotides is not interrupted. The genomic blot analysis of human placenta DNA indicates the presence of multiple LDH-A gene-related sequences.
Intracellular distribution of haem after uptake by different receptors. Haem-haemopexin and haem-asialo-haemopexin
Authors: Smith A
Published: August 2015

How the interaction of haemopexin with two different receptors affects its subsequent metabolism and ‘intracellular’ haem transport was examined by using mesohaem-haemopexin and mesohaem-asialo-haemopexin. The physical properties of the two haem proteins, including their absorption and c.d. spectra, are similar. Binding studies in vitro showed that haem-asialo-haemopexin interacts with both the haemopexin-specific and galactose-specific receptors on liver plasma membranes, but that haem-haemopexin interacts only with the haemopexin receptor. In vivo haem-asialo-haemopexin rapidly interacts with the liver via the galactose-specific receptor, since the protein is extensively catabolized and uptake is blocked by asialofetuin. Haem iron from haem-asialo-haemopexin is not accumulated in the liver to the same extent as from intact haem-haemopexin, and the native sialylated protein is not proteolysed. Moreover, after fractionation of homogenized liver by using colloidal-silica gradients, liver-associated haem-haemopexin and haem-asialo-haemopexin produced distinctly different patterns for both protein and ligand, consistent with their uptake by two distinct receptors. These results demonstrate that the interaction of haemopexin with different receptors influences its subsequent metabolic fate and that haem iron from haem-haemopexin is efficiently conserved only if it enters the liver cell via the specific haemopexin receptor.
Prolinase and non-specific dipeptidase of human kidney
Authors: Priestman D A , Butterworth J
Published: August 2015

Human kidney prolinase, assayed with Pro-Ala, and non-specific dipeptidase, assayed with Gly-Leu, were purified by using DEAE-cellulose, gel-filtration, metal-ion-chelate, hydrophobic and adsorption chromatography and chromatofocusing. Both enzymes gave single peaks of activity that were congruent and the ratio of their activities was constant throughout the purification. Gel filtration indicated an Mr of 100 000 and chromatofocusing a pI of 5.4. Ni2+-chelate chromatography demonstrated the presence of exposed histidine residues on the enzyme and was an effective separative procedure. Polyacrylamide-gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. Both enzyme activities decayed at the same rate at 53 degrees C and were inhibited to the same extent by p-hydroxymercuribenzoate. Of six non-specific dipeptidase substrates tested Gly-Leu gave the highest activity, and of six prolinase substrates Pro-Leu had the highest activity. Gly-Leu was hydrolysed at double the rate of Pro-Leu. Pro-Ala was a competitive inhibitor of activity towards Gly-Leu, and Gly-Leu was a competitive inhibitor of activity towards Pro-Ala. Mixed-substrate studies strongly suggested that Gly-Leu and Pro-Ala were hydrolysed at a common active site. The data are consistent with prolinase and non-specific dipeptidase activity in human kidney being due to a single enzyme.
Fuel utilization in colonocytes of the rat
Authors: Ardawi M S M , Newsholme E A
Published: August 2015

In incubated colonocytes isolated from rat colons, the rates of utilization O2, glucose or glutamine were linear with respect to time for over 30 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 30 min. Glutamine, n-butyrate or ketone bodies were the only substrates that caused increases in O2 consumption by isolated incubated colonocytes. The maximum activity of hexokinase in colonic mucosa is similar to that of 6-phosphofructokinase. Starvation of the donor animal decreased the activities of hexokinase and 6-phosphofructokinase, whereas it increased those of glucose-6-phosphatase and fructose-bisphosphatase. Isolated incubated colonocytes utilized glucose at about 6.8 mumol/min per g dry wt., with lactate accounting for 83% of glucose removed. These rates were not affected by the addition of glutamine, acetoacetate or n-butyrate, and starvation of the donor animal. Isolated incubated colonocytes utilized glutamine at about 5.5 mumol/min per g dry wt., which is about 21% of the maximum activity of glutaminase. The major end-products of glutamine metabolism were glutamate, aspartate, alanine and ammonia. Starvation of the donor animal decreased the rate of glutamine utilization by colonocytes, which is accompanied by a decrease in glutamate formation and in the maximum activity of glutaminase. Isolated incubated colonocytes utilized acetoacetate at about 3.5 mumol/min per g dry wt. This rate was not markedly affected by addition of glucose or by starvation of the donor animal. When colonocytes were incubated with n-butyrate, both acetoacetate and 3-hydroxybutyrate were formed, with the latter accounting for only about 19% of total ketones produced.
Decanoyl lysophosphatidic acid induces platelet aggregation through an extracellular action. Evidence against a second messenger role for lysophosphatidic acid
Authors: Watson S P , McConnell R T , Lapetina E G
Published: August 2015

Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into its corresponding phosphatidic acid and lysophosphatidic acid derivatives, thereby providing a means of introducing these two compounds into platelets. 1-Decanoyl-2-lyso-3-sn-phosphatidic acid, when added directly to platelets, induced platelet aggregation and raised intracellular Ca2+ levels at concentrations of 0.3 microM upwards, but was without effect when formed intracellularly from 1,2-didecanoylglycerol at an estimated concentration of approx. 47 microM. This indicates that the site of platelet activation by lysophosphatidic acid is extracellular. A concentration of thrombin (0.2 unit/ml), which produced maximal platelet aggregation, caused an estimated intracellular formation of 20 microM-lysophosphatidic acid in the presence of 2 mM-Ca2+; however, there was no detectable release of lysophosphatidic acid into the bathing medium. Lysophosphatidic acid, therefore, may not be an intracellular second messenger involved in platelet aggregation by thrombin.
Biosynthesis of complement components by cultured rat hepatocytes
Authors: Anthony R , Morrison L , MacSween R N , Whaley K
Published: August 2015

Hepatocytes which had been isolated from the livers of Charles River rats were cultured in vitro. The cells were shown to synthesize albumin and the complement components C4, C2, C3 and B. Pulse-label studies with [35S]methionine showed that C4 and C3 were synthesized as single polypeptide chains. Pro-C4 did not appear to be converted into the plasma form of C4 intracellularly, whereas cell lysates contained the α- and β-chains of plasma C3 as well as pro-C3. It is concluded that culture of rat hepatocytes in vitro provides a useful technique for studies of the synthesis of complement components.
Cloning and nucleotide sequence analysis of transfer RNA genes from Mycoplasma mycoides
Authors: Samuelsson T , Elias P , Lustig F , Guindy Y S
Published: August 2015

As part of an investigation of the tRNA genes of Mycoplasma mycoides, two HindIII fragments of mycoplasma DNA comprising 0.4 and 2.5 kilobases (kb), respectively, were cloned in pBR322 and their nucleotide sequences determined. Only one tRNA gene was found in the 0.4 kb fragment, the gene for tRNAArg with the anticodon TCT, while the 2.5 kb fragment contained nine different tRNA genes arranged in a cluster which presumably constitutes a transcriptional unit. The clustered tRNA genes, with their respective anticodons, were as follows: Arg (ACG), Pro (TGG), Ala (TGC), Met (CAT), Ile (CAT), Ser (TGA), fMet (CAT), Asp (GTC), and Phe (GAA).
Characterization of triacylglycerol hydrolase activities in isolated myocardial cells from rat heart
Authors: Ramírez I , Kryski A J , Ben-Zeev O , Schotz M C , Severson D L
Published: August 2015

Triacylglycerol (TG) hydrolase activities were characterized in myocytes isolated from rat hearts. Acid hydrolase activity with a pH optimum of 5 could be measured in myocyte homogenates, and the subcellular distribution suggested that this activity originated in lysosomes. Lipoprotein lipase (LPL) was also present in myocyte homogenates, as evidenced by TG hydrolase activity that was stimulated by serum and apolipoprotein CII, and inhibited by apolipoprotein CIII2, high ionic strength (NaCl and MgCl2, I = 1 M) and antibodies to LPL. Serum-independent neutral (pH 7.5) TG hydrolase activity was less sensitive to inhibition by 1 M-NaCl, by antibodies to LPL and by preincubation at 40 degrees C than was serum-stimulated hydrolase activity. Furthermore, there were modest but significant differences in the subcellular distribution of the serum-independent and serum-stimulated hydrolase activities. Hydrolase activities in myocyte homogenates could be solubilized by 7.2 mM-deoxycholate. Acid hydrolase activity was recovered in the unbound fraction after heparin-Sepharose chromatography, whereas LPL was bound to the affinity column and was eluted by 0.9-1.2 M-NaCl. Approximately one-third of the serum-independent TG hydrolase activity was not bound to the heparin-Sepharose affinity column. This unbound TG hydrolase activity had a pH optimum of 7 and was stimulated by 50 mM-MgCl2, but not by serum and was resistant to inhibition by high ionic strength (1 M-NaCl), to preincubation at 40 degrees C for 2 h, and by antibodies to LPL. It is concluded that, in addition to an acid lysosomal TG hydrolase and LPL, myocytes from rat heart contain a serum-independent TG hydrolase with unique characteristics.
Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells
Authors: Burgess G M , McKinney J S , Irvine R F , Putney J W
Published: August 2015

The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.
Regulation of storage-protein synthesis in pea (Pisum sativum L.) cotyledons under conditions of sulphur deficiency
Authors: Evans I M , Gatehouse J A , Boulter D
Published: August 2015

The effects of sulphur deficiency on the expression of storage-protein genes in developing pea (Pisum sativum) cotyledons were studied. Legumin-gene transcription was decreased by S-deficiency, but not to the same extent as the decrease in the level of legumin mRNA. Vicilin-gene transcription was not significantly affected. Control of gene expression may thus occur during transcription and/or post-transcriptional events.
Vitamin A effects on UMR 106 osteosarcoma cells are not mediated by specific cytosolic receptors
Authors: Oreffo R O , Francis J A , Triffitt J T
Published: August 2015

Retinol and retinoic acid at 20 microM altered cell morphology and inhibited cell proliferation of UMR 106 osteosarcoma cells in culture. No specific cytosolic binding proteins for retinol could be detected.
Involvement of calcium-sensitive phospholipid-dependent protein kinase in control of acid secretion by isolated rat parietal cells
Authors: Anderson N G , Hanson P J
Published: August 2015

The relative potency with which phorbol esters inhibited histamine-stimulated aminopyrine accumulation (an index of acid secretion) paralleled that which has been established for the activation of purified protein kinase C. The inhibitory effect of 1-oleoyl-2-acetylglycerol on aminopyrine accumulation stimulated by various secretagogues was similar to that of 12-O-tetradecanoylphorbol 13-acetate. Protein kinase C activity was present in a parietal-cell-enriched fraction. In conclusion, protein kinase C could be involved in mechanisms regulating gastric acid secretion.
Regulation of retinal transducin by C-terminal peptides of rhodopsin
Authors: Takemoto D J , Takemoto L J , Hansen J , Morrison D
Published: August 2015

Transducin is a multi-subunit guanine-nucleotide-binding protein that mediates signal coupling between rhodopsin and cyclic GMP phosphodiesterase in retinal rod outer segments. Whereas the T alpha subunit of transducin binds guanine nucleotides and is the activator of the phosphodiesterase, the T beta gamma subunit may function to link physically T alpha with photolysed rhodopsin. In order to determine the binding sites of rhodopsin to transducin, we have synthesized eight peptides (Rhod-1 etc.) that correspond to the C-terminal regions of rhodopsin and to several external and one internal loop region. These peptides were tested for their inhibition of restored GTPase activity of purified transducin reconstituted into depleted rod-outer-segment disc membranes. A marked inhibition of GTPase activity was observed when transducin was pre-incubated with peptides Rhod-1, Rhod-2 and Rhod-3. These peptides correspond to opsin amino acid residues 332-339, 324-331 and 317-321 respectively. Peptides corresponding to the three external loop regions or to the C-terminal residues 341-348 did not inhibit reconsituted GTPase activity. Likewise, Rhod-8, a peptide corresponding to an internal loop region of rhodopsin, did not inhibit GTPase activity. These findings support the concept that these specific regions of the C-terminus of rhodopsin serve as recognition sites for transducin.
Isolation and characterization of two sialoproteins present only in bone calcified matrix
Authors: Franzén A , Heinegård D
Published: August 2015

Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.
Different forms of human liver glutathione s-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits
Authors: Singh S V , Dao D D , Partridge C A , Theodore C , Srivastava S K , Awasthi Y C
Published: August 2015

Four immunologically distinct subunits were characterized in glutathione (GSH) S-transferases of human liver. Five cationic enzymes (pI 8.9, 8.5, 8.3, 8.2 and 8.0) have an apparently similar subunit composition, and are dimers of 26 500-Mr (A) and 24 500-Mr (B) subunits. A neutral enzyme, pI 6.8, is a dimer of B-type subunits. One of the anionic enzymes, pI 5.5, is also a dimer of 26 500-Mr subunits. However, the 26 500-Mr subunits of this anionic enzyme form are immunologically distinct from the A subunits of the cationic enzymes, and have been designated as A'. Immunoabsorption studies with the neutral enzyme, BB, and the antibodies raised against the cationic enzymes (AB) indicate that A and B subunits are immunologically distinct. Hybridization in vitro of the A and B subunits of the cationic enzymes (AB) results in the expected binary combinations of AA, AB and BB. Studies with the hybridized enzyme forms indicate that only the A subunits express GSH peroxidase activity. A' subunits have maximum affinity for p-nitrobenzyl chloride and p-nitrophenyl acetate, and the B subunits have highest activity towards 1-chloro-2,4-dinitrobenzene. The other anionic form, pI 4.5, present in liver is a heterodimer of 22 500-Mr (C) and B subunits. The C subunits of this enzyme are probably the same as the 22 500-Mr subunits present in human lung and placental GSH transferases. The distinct immunological nature of B and C subunits was also demonstrated by immunoaffinity and subunit-hybridization studies. The results of two-dimensional polyacrylamide-gel-electrophoretic analyses indicate that in human liver GSH transferases, three charge isomers of Mr 26 500 (A type), two charge isomers of Mr 24 500 (B type) and two charge isomers of Mr 22 500 (C type) subunits are present.
The origin of human cartilage proteoglycan link-protein heterogeneity and fragmentation during aging
Authors: Mort J S , Caterson B , Poole A R , Roughley P J
Published: August 2015

Human articular-cartilage link proteins are resolved into three components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicative of three different structures. The action of the proteinase clostripain yields a single link-protein component with electrophoretic properties analogous to those of the smallest (most mobile) native link protein, suggesting that this link protein may be derived naturally from one or both of the larger molecules by proteolytic cleavage in situ. Upon chemical deglycosylation of native link protein two components are resolved, suggesting that two of the link proteins differ only in their degree and/or type of oligosaccharide substitution. This pattern is compatible with a proteolytic origin for the smallest link protein. During aging further proteolytic fragmentation occurs, though it is only apparent on reduction of disulphide bonds. This fragmentation occurs at identical sites in all three native link proteins, indicating the existence of a large region common to all the link proteins, which appears to consist predominantly of the C-terminal half of the molecules. These observations are compatible with the variation in oligosaccharide and proteolytic heterogeneity occurring at the N-terminus of the link proteins.
The effect of metabolic acidosis on the synthesis and turnover of rat renal phosphate-dependent glutaminase
Authors: Tong J , Harrison G , Curthoys N P
Published: August 2015

Regulation of the mitochondrial phosphate-dependent glutaminase activity is an essential component in the control of renal ammoniagenesis. Alterations in acid-base balance significantly affect the amount of the glutaminase that is present in rat kidney, but not in brain or small intestine. The relative rates of glutaminase synthesis were determined by comparing the amount of [35S]methionine incorporated into specific immunoprecipitates with that incorporated into total protein. In a normal animal, the rate of glutaminase synthesis constitutes 0.04% of the total protein synthesis. After 7 days of metabolic acidosis, the renal glutaminase activity is increased to a value that is 5-fold greater than normal. During onset of acidosis, the relative rate of synthesis increases more rapidly than the appearance of increased glutaminase activity. The increased rate of synthesis reaches a plateau within 5 days at a value that is 5.3-fold greater than normal. Recovery from chronic acidosis causes a rapid decrease in the relative rate of glutaminase synthesis, but a gradual decrease in glutaminase activity. The former returns to normal within 2 days, whereas the latter requires 11 days. The apparent half-time for glutaminase degradation was found to be 5.1 days and 4.7 days for normal and acidotic rats respectively. These results indicate that the increase in renal glutaminase activity associated with metabolic acidosis is due primarily to an increase in its rate of synthesis. From the decrease in activity that occurs upon recovery from acidosis, the true half-life for the glutaminase was estimated to be 3 days.
Glucose utilization in vivo and insulin-sensitivity of rat brown adipose tissue in various physiological and pathological conditions
Authors: Ferré P , Burnol A F , Leturque A , Terretaz J , Penicaud L , Jeanrenaud B , Girard J
Published: August 2015

Brown-adipose-tissue glucose utilization rate and its insulin-sensitivity were measured in vivo in the anaesthetized rat by a 2-deoxy[1-3H]glucose technique. Glucose utilization can be increased 60-fold by insulin, to reach extremely high rates. Glucose utilization and its insulin-sensitivity are modulated in accordance with physiological or pathological conditions.
Enhancement of glucagon secretion from isolated rat islets of Langerhans by phorbol 12-myristate 13-acetate
Authors: Hii C S , Stutchfield J , Howell S L
Published: August 2015

The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA), at concentrations of 0.1 microM and above, stimulated secretion of glucagon and of insulin from isolated rat islets of Langerhans incubated in the presence of 5.5 mM-glucose. Stimulation of secretion of both hormones by 1 microM-PMA persisted in the absence of external Ca2+, and could be abolished by incubating the islets at 4 degrees C. These findings suggest a role of protein kinase C in the alpha-cell (and beta-cell) secretory mechanism.
Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells
Authors: Habberfield A D , Dix C J , Cooke B A
Published: August 2015

A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature-sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone.
Inhibition of hepatic and extrahepatic glutathione S-transferases by primary and secondary bile acids
Authors: Hayes J D , Mantle T J
Published: August 2015

Glutathione S-transferases are a complex family of dimeric proteins that play a dual role in cellular detoxification; they catalyse the first step in the synthesis of mercapturic acids, and they bind potentially harmful non-substrate ligands. Bile acids are quantitatively the major group of ligands encountered by the glutathione S-transferases. The enzymes from rat liver comprise Yk (Mr 25 000), Ya (Mr 25 500), Yn (Mr 26 500), Yb1, Yb2 (both Mr 27 000) and Yc (Mr 28 500) monomers. Although bile acids inhibited the catalytic activity of all transferases studied, the concentration of a particular bile acid required to produce 50% inhibition (I50) varies considerably. A comparison of the I50 values obtained with lithocholate (monohydroxylated), chenodeoxycholate (dihydroxylated) and cholate (trihydroxylated) showed that, in contrast with all other transferase monomers, the Ya subunit possesses a relatively hydrophobic bile-acid-binding site. The I50 values obtained with lithocholate and lithocholate 3-sulphate showed that only the Ya subunit is inhibited more effectively by lithocholate than by its sulphate ester. Other subunits (Yk, Yn, Yb1 and Yb2) were inhibited more by lithocholate 3-sulphate than by lithocholate, indicating the existence of a significant ionic interaction, in the bile-acid-binding domain, between (an) amino acid residue(s) and the steroid ring A. By contrast, increasing the assay pH from 6.0 to 7.5 decreased the inhibitory effect of all bile acids studied, suggesting that there is little significant ionic interaction between transferase subunits and the carboxy group of bile acids. Under alkaline conditions, low concentrations (sub-micellar) of nonsulphated bile acids activated Yb1, Yb2 and Yc subunits but not Yk, Ya and Yn subunits. The diverse effects of the various bile acids studied on transferase activity enables these ligands to be used to help establish the quaternary structure of individual enzymes. Since these inhibitors can discriminate between transferases that appear to be immunochemically identical (e.g. transferases F and L), bile acids can provide information about the subunit composition of forms that cannot otherwise be distinguished.
Cucurbitacin Δ23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions
Authors: Dirr H W , Schabort J C , Weitz C
Published: August 2015

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 × 10(-7) cm2 × s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.
Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver
Authors: Hayes J D
Published: August 2015

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].
Monoclonal antibodies to different protein-related epitopes of human articular cartilage proteoglycans
Authors: Glant T T , Mikecz K , Poole A R
Published: August 2015

Monoclonal antibodies produced against chondroitinase-treated human adult cartilage proteoglycans were selected for their ability to recognize epitopes on native proteoglycans. Binding analyses revealed that four of these monoclonal antibodies (BCD-4, BCD-7, EFG-4 and KPC-190) each recognized a different epitope on the same proteoglycan molecule which represents a subpopulation of a high buoyant density (D1) fraction of human articular cartilage proteoglycans (10, 30, 50 and 60% in fetal-newborn, 1.5 years old, 15 years old and 52-56 years old cartilages, respectively). Analysis of epitope specificities revealed that BCD-7 and EFG-4 monoclonal antibodies recognized epitopes on proteoglycan monomer which are associated with the protein structure in that they are sensitive to cleavage by Pronase, papain and alkali treatment and do not include keratan sulphate, chondroitin sulphate or oligosaccharides. The BCD-4 and KPC-190 epitopes also proved to be sensitive to Pronase or papain digestion or to alkali treatment, but keratanase or endo-beta-galactosidase also reduced the immunoreactivity of these epitopes. These observations indicate that the BCD-4 and KPC-190 epitopes represent peptides substituted with keratan sulphate or keratan sulphate-like structures. The BCD-4 epitope is, however, absent from a keratan sulphate-rich fragment of human adult proteoglycan, while the other three epitopes were detected in this fragment. None of these four epitopes were detected in the link proteins of human cartilage, in the hyaluronic acid-binding region of human newborn cartilage proteoglycan, in Swarm rat chondrosarcoma proteoglycan, in chicken limb bud proteoglycan monomer and in the small dermatan sulphate-proteoglycan of bovine costal cartilage. EFG-4 and KPC-190 epitopes were not detected in human fetal cartilage proteoglycans, although fetal molecules contained trace amounts of epitopes reactive with BCD-4 and BCD-7 antibodies.
Role of phospholipase C and protein kinase C in vasoconstrictor-induced prostaglandin synthesis in cultured rat renal mesangial cells
Authors: Pfeilschifter J , Kurtz A , Bauer C
Published: August 2015

It was the aim of the present study to find out if a common mechanism exists by which the vasoconstrictive hormones angiotension II, noradrenaline and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) increase prostaglandin E2 (PGE2) synthesis in cultures of rat renal mesangial cells. Angiotension II, noradrenaline and AGEPC stimulated PGE2 synthesis and uptake of 45Ca2+ in cultured mesangial cells. Both of these effects could be completely suppressed by the calcium channel blocker verapamil. Angiotensin II, noradrenaline and AGEPC caused a rapid breakdown of phosphatidylinositol 4,5-bisphosphate with a concomitant increase of 1,2-diacylglycerol and inositol trisphosphate, indicating an activation of phospholipase C by these hormones. Addition of verapamil had no effect on the hormone-induced stimulation of phospholipase C. The synthetic analogue of diacylglycerol, 1-oleoyl-2-acetylglycerol, and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), both of which are known to stimulate protein kinase C, enhanced PGE2 synthesis. Chelation of extracellular calcium with EDTA or addition of verapamil abolished the effect of 1-oleoyl-2-acetylglycerol and phorbol ester on PGE2 synthesis. 1-Oleoyl-2-acetylglycerol and phorbol ester increased the uptake of 45Ca2+ by the cells in a dose-dependent manner and this effect could be blocked by verapamil. The entirety of these data leads us to suggest that vasoconstrictor-evoked synthesis of PGE2 in rat mesangial cells is mediated by the subsequent activation of phospholipase C and protein kinase C. The activation of protein kinase C by diacylglycerol is likely to be involved in the increase of the calcium permeability of the plasma membrane which is a prerequisite for PGE2 synthesis induced by vasoconstrictive hormones.
A reassessment of the assay for the asialoglycoprotein receptor and its use in the quantification of receptor distribution in hepatocytes
Authors: Grant D A W , Kaderbhai N
Published: August 2015

The assay for the fucose-binding protein described by Lehrman & Hill [(1983) Methods Enzymol. 98, 309-319] was adapted for the measurement of the asialoglycoprotein receptor in rat liver. The amount of ligand bound to the plasma-membrane-associated or affinity-purified receptor was acutely sensitive to the concentrations of Triton X-100 and NaCl in the assay: 0.02% (v/v) Triton X-100 increased ligand binding to the two preparations by 100% and 40% respectively. Higher concentrations of detergent progressively decreased binding, and in 0.32% Triton X-100 it was about 30% of the value obtained in detergent-free buffer. The addition of increasing concentrations of NaCl to the assay progressively inhibited ligand binding to the membrane-associated receptor, whereas there was a 60% increase in binding to the pure receptor in the presence of 0.1-0.2 M-NaCl. These effects could not be identified in the original assay procedure described by Hudgin, Pricer, Ashwell, Stockert & Morell [(1974) J. Biol. Chem. 249, 5536-5543]. Using optimal assay conditions the binding of 125I-beta-D-galactosyl-bovine serum albumin to both the membrane-associated and purified receptor was inhibited by 50% by 1 nM-beta-D-galactosyl-bovine serum albumin and -asialoorosomucoid and by approx. 100 microM-beta-L-fucosyl-bovine serum albumin, whereas beta-D-galactose, lactose and beta-L-fucose had no effect on ligand binding up to concentrations of 1 mM, 500 microM and 5 mM respectively. KD values of 0.94 and 1.25 nM and Bmax. values of 40 and 1660 pmol of D-galactosyl-bovine serum albumin bound/mg of receptor were obtained for the membrane-bound and purified receptor respectively. Hill-plot analysis of the same data gave slopes of 0.96 and 1.01. Scatchard analysis of saturation-binding studies with other subcellular fractions indicated that the receptor was distributed in the proportions 72:23:2.5:2.5 between total microsomal fractions, plasma membrane, Golgi and canalicular membrane respectively. The receptor was about 1% of the total protein in each compartment and was estimated to be about 0.3% of the total liver protein.
A study of the kinetics of the reaction of ligands with the liganded states of mouse embryonic haemoglobins
Authors: Brittain T , Sutherland J , Greenwood C
Published: August 2015

The reactivities of the liganded states of the embryonic haemoglobins of the mouse with both O2 and CO were measured and compared with the reactivities of the adult protein. Laser-photolysis experiments on the recombination of O2 with the partially oxygenated proteins indicates chain heterogeneity in the adult and embryonic EII and EIII species, with the difference in subunit reactivity being greatest in the embryonic species. Haemoglobin EI shows chain equivalence in these experiments. The homogeneous time courses observed for the O2-dissociation reactions are consistent with chain equivalence within all the proteins with regard to this reaction. The specific values obtained for the respective rate constants from each of these studies indicates that the high O2 affinity previously reported for haemoglobin EI is, in greatest part, due to its low O2 dissociation rate. Flash-photolysis studies on the binding of CO with the partially liganded forms of the proteins show the same patterns of chain heterogeneity as seen in O2-binding studies.
Evidence that lysosomes are not involved in the degradation of myofibrillar proteins in rat skeletal muscle
Authors: Lowell B B , Ruderman N B , Goodman M N
Published: August 2015

To examine the role of lysosomes in the degradation of skeletal-muscle myofibrillar proteins, we measured the release of N tau-methylhistidine from perfused muscle of starved and fed rats in the presence or absence of agents that inhibit lysosomal proteinase activity. After 1 day of starvation, the release of N tau-methylhistidine by perfused muscle of 4-, 8- and 24-week-old rats increased by 322, 159 and 134% respectively. On the other hand, total protein breakdown, assessed by tyrosine release, increased by 62, 20 and 20% respectively. Inhibitors of lysosomal proteinases as well as high concentrations of insulin or amino acids failed to diminish the release of N tau-methylhistidine by perfused muscle of starved and fed rats, despite a 25-35% inhibition of total protein breakdown. The data strongly suggest that the complete breakdown of myofibrillar proteins occurs via a non-lysosomal pathway. They also suggest that total proteolysis, which primarily reflects non-myofibrillar protein breakdown, occurs at least in part within lysosomes.
Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet
Authors: Harris R A , Paxton R , Goodwin G W , Powell S M
Published: August 2015

Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.
Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism
Authors: Paxton R , Scislowski P W , Davis E J , Harris R A
Published: August 2015

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.
The effect of GTP on inositol 1,4,5-trisphosphate-stimulated Ca2+ efflux from a rat liver microsomal fraction. Is a GTP-dependent protein phosphorylation involved?
Authors: Dawson A P , Comerford J G , Fulton D V
Published: August 2015

GTP, when added to a rat liver microsomal fraction that had previously been allowed to accumulate Ca2+, causes a slow release of Ca2+, which is greatly enhanced by addition of inositol trisphosphate (IP3). The Ca2+ release caused by IP3 under these conditions is very much greater than that observed in the absence of GTP. The effect of GTP is dependent on the presence of polyethylene glycol in the incubation medium and is not due to inhibition of the Ca2+-accumulation system. The response to GTP is time-dependent, particularly at low (4 microM) GTP concentrations, and cannot be mimicked by ATP, ITP, CTP, UTP and GDP. Studies with [gamma-32P]GTP show that, during incubation with microsomal fractions, the terminal phosphate of GTP is transferred to two protein species, of Mr 38 000 and 17 000. These protein phosphorylations are still present when an excess of unlabelled ATP is included in the incubation mixture, but appear to be unaffected by the presence or absence of IP3 and polyethylene glycol. As a working hypothesis, it is suggested that a protein, phosphorylated by GTP, has to bind to the microsomal membranes before IP3 can stimulate Ca2+ release, and that, in vitro, the binding of this protein is favoured by the presence of polyethylene glycol.