Cholate-independent retinyl ester hydrolysis. Stimulation by Apo-cellular retinol-binding protein

Apo-cellular retinol-binding protein (apoCRBP) activated the hydrolysis of endogenous retinyl esters in rat liver microsomes by a cholate independent retinyl ester hydrolase. A Michaelis-Menten relationship was observed between the apoCRBP concentration and the rate of retinol formation, with half-maximum stimulation at 2.6 +/- 0.6 microM (mean +/- S.D., n = 5). Two other retinol-binding proteins, bovine serum albumin and beta-lactoglobulin, acceptors for the rapid and spontaneous hydration of retinol from membranes, had no effect up to 90 microM. These data suggest activation of the hydrolase by apoCRBP directly, rather than by facilitating removal of retinol from membranes. The hydrolase responding was the cholate-independent/cholate-inhibited retinyl ester hydrolase as shown by: 60% inhibition of the apoCRBP effect by 3 mM cholate; apoCRBP enhancement of retinyl ester hydrolysis in liver microsomes that had no detectable cholate-enhanced activity; inhibition of cholate-dependent, but not apoCRBP-stimulated retinyl ester hydrolysis by rabbit anti-rat cholesteryl esterase. Compared to the rate (mean +/- S.D. of [n] different preparations) supported by 5 microM apoCRBP in liver microsomes of 6.7 +/- 3.7 pmol/min/mg protein [10], microsomes from rat lung, kidney, and testes had endogenous retinyl ester hydrolysis rates of 1.8 +/- 0.3 [5], 0.5 +/- 0.2 [3], and 0.3 +/- 0.2 [5] pmol/min/mg protein, respectively. N-Ethylmaleimide and N-tosyl-L-phenylalanine chloromethyl ketone were potent inhibitors of apoCRBP-stimulated hydrolysis with IC50 values of 0.25 and 0.15 mM, respectively, but phenylmethylsulfonyl fluoride and diisopropyl-fluorophosphate were less effective with IC50 values of 1 mM, indicating the importance of imidazole and sulfhydryl groups to the activity. These data provide evidence of a physiological role for the cholate-independent hydrolase in retinoid metabolism and suggest that apoCRBP is a signal for retinyl ester mobilization.     

Topology of membrane exposure in the renal cortex slice. Studies of glutathione and maltose cleavage

We measured glycine release from ([2-3H]glycine)-labelled GSH and glucose formation from maltose incubated with rat kidney whole cortex homogenate, thin cortex slices or collagenase-treated tubule fragments. Liberation of glycine was inhibited (74-83%) by serine borate (20 mM), indicating a gamma-glutamyltransferase-dependent hydrolysis of GSH. In whole cortex homogenate, the GSH cleavage activity was 17.4 +/- 0.6 nmol GSH degraded/mg protein per min (mean +/- S.D.); cleavage activity by intact slices was 3.5 +/- 0.7 (P less than 0.001 relative to whole cortex homogenate) and in tubule fragments 9.4 +/- 0.8 (P less than 0.001). Homogenizing the tissue preparation increased cleavage rate in slices about 4-fold (12.4 +/- 2.9; P less than 0.005 relative to intact slice) but did not change the rate in tubule fragments (9.8 +/- 0.5). Maltose cleavage activity in whole cortex homogenate was 512 +/- 22 nmol glucose formed/mg protein per min, in slices 162 +/- 12, and in tubules 884 +/- 48. These findings imply that substrate in the incubation medium has a limited access to the luminal membrane of cortex slices but not of tubule fragments. They further imply that basolateral membrane is preferentially exposed in the slice preparation.     

A N-methyltransferase in human brain catalyses N-methylation of 1,2,3,4-tetrahydroisoquinoline into N-methyl-1,2,3,4-tetrahydroisoquinoline, a precursor of a dopaminergic neurotoxin, N-methylisoquinolinium ion

N-methylation of 1,2,3,4-tetrahydroisoquinoline (TIQ) present in human brain was found by a N-methyltransferase in human brain homogenate. Formation of N-methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) from TIQ was quantitatively assayed by high-performance liquid chromatography with electrochemical detection. The reaction required S-adenosyl-L-methionine (SAM) as a methyl donor and in terms of SAM the value of the Michaelis constant, Km, and of the maximal velocity, Vmax, were 5.11 +/- 1.69 microM and 7.31 +/- 0.21 pmol/min/mg protein, respectively. The value of Km and Vmax in terms of TIQ were 20.9 +/- 5.5 microM and 7.98 +/- 1.21 pmol/min/mg protein, respectively. The optimal pH of the reaction was 8.25. A major part of the N-methyltransferase activity was found in the cytosolic fraction of human cortex. Enzymatic formation of NMTIQ indicates that in human brain this compound may be an intermediate of biosynthesis of a potent neurotoxin of dopamine metabolism, N-methylisoquinolinium ion, from naturally-occurring TIQ.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.     

Precursor utilization of 5-hydroxytryptophan for 5-hydroxytryptamine biosynthesis in isolated and perfused rabbit and rat lungs

The present study was designed to investigate whether lungs can utilize 5-hydroxytryptophan (5-HTP), formed elsewhere and transported, for the synthesis of 5-hydroxytryptamine (5-HT). [14C]5-HTP uptake was 7.7 +/- 1.1 and 3.9 +/- 0.2% by rabbit and rat lungs, respectively, after 1 h of perfusion with 10 microM [14C]5-HTP. There was an increase in the lung uptake of [14C]5-HTP when the lungs were preperfused with 0.5 mM chlorphentermine (CP) and the uptake was low when the lungs were preperfused with 0.1 mM hydroxybenzylhydrazine dihydrochloride (HBH). The perfusate concentration of 5-hydroxyindole acetic acid (5-HIAA) increased significantly (3-4 micrograms/100 mL) during rabbit lung perfusion with 10 microM [14C]5-HTP and this did not change significantly when the lungs were preperfused with 0.5 mM CP. However, 5-HT increased with time in the perfusate. 5-HT, but not 5-HIAA, was detected in the perfusate and increased with time of perfusion when the rat lungs were perfused either with 10 microM 5-HTP or with 0.5 mM CP and 10 microM 5-HTP. However, no metabolites were detected in either the rabbit lung or rat lung perfusates when they were preperfused with 0.1 mM HBH. Lung contents of 5-HT and 5-HIAA were significantly higher in the rat lungs and only 5-HIAA increased in rabbit lungs after 1 h of perfusion with 10 microM 5-HTP. Preperfusion with 0.5 mM CP resulted in a greater increase in the 5-HT content of both rabbit and rat lungs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of aminopeptidase activity in scab mites (Psoroptes spp.)

Soluble and membrane-bound aminopeptidase activities were demonstrated in extracts of P. cuniculi (Delafond). Leucine aminopeptidase (LAP) activity in the soluble fraction of P. cuniculi extracts displayed substrate preference for amino acid derivatives with terminal leucine and methionine over those with acidic, basic or heterocyclic groups. P. cuniculi LAP was inhibited by leucinethiol (IC(50) = 1.4 +/- 0.4 nM), bestatin (IC(50) = 3.9 +/- 1.7 microM), Arphamenine A (IC(50) = 0.37 +/- 0.03 mM) the chelating agent 1,10-phenanthroline (IC(50) = 2.3 +/- 0.5 mM), Zn(2+), Cu(2+) Ni(2+), and Co(2+), and activated by Mn(2+) and Mg(2+). The LAP activity was visualised as a single major band after electrophoresis on native gels and eluted from a size exclusion column as a single major peak representing a molecular mass range of 85-116 kDa. Degenerate oligonucleotide primers were used to amplify short fragments of genomic DNA containing nucleotide sequence coding for the cation-binding motifs of the co-catalytic Zn(2+) binding domains of dizinc leucine aminopeptidases in both P. cuniculi and P.ovis (Hering). The major soluble aminopeptidase from these mites therefore displays most of the characteristics associated with typical cytosolic leucine aminopeptidases belonging to the M17 family of metalloproteinases.     

Pharmacological modulation of fluid secretion in the pigmented rabbit conjunctiva

We determined net fluid secretion rate across the pigmented rabbit conjunctiva in the presence and absence of pharmacological agents known to affect active Cl- secretion and Na+ absorption. Fluid flow across a freshly excised pigmented rabbit conjunctiva mounted between two Lucite half chambers was measured by a pair of capacitance probes in an enclosed cabinet maintained at 37 degrees C and a relative humidity of 70%. Fluid transport was also measured in the presence of compounds known to affect active Cl- secretion (cAMP, UTP, and ouabain), Na+ absorption (D-glucose), or under the Cl--free condition on both sides of the tissue. Net fluid secretion rate across the pigmented rabbit conjunctiva in the serosal-to-mucosal direction at baseline was 4.3+/-0.2 microl/hr/cm2 (mean +/- s.e.m.). Net fluid secretion rate was increased approximately two-fold by mucosally applied 1 mM 8-Br cAMP (8.4+/-0.4 microl/hr/cm2) and 10 microM UTP (9.8+/-0.6 microl/hr/cm2), but was abolished by either serosally applied 0.5 mM ouabain (0.3+/-0.1 microl/hr/cm2) or under the Cl--free conditions (0.06+/-0.04 microl/hr/cm2). Mucosal addition of 20 mM D-glucose decreased net fluid secretion rate to 1.0+/-0.5 microl/hr/cm2. In conclusion, the pigmented rabbit conjunctiva appears to secrete fluid secondary to active Cl- secretion. This net fluid secretion is subject to modulation by changes in active Cl- secretion rate and in mucosal fluid composition such as glucose concentration.     

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Rat Small Intestine

Glucose-6-phosphate dehydrogenase (G6PD) was purified from rat small intestine with 19.2% yield and had a specific activity of 53.8 units per miligram protein. The pH optimum was determined to be 8.1. The purified rat small intestinal G6PD gave one activity, one protein band on native PAGE. The observation of one band on SDS/PAGE with an Mr of 48 kDa and a specific activity lower than expected may suggest the proteolytically affected enzyme or different form of G6PD in the rat small intestine. The activation energy, activation enthalpy, Q10, and optimum temperature from Arrhenius plot for the rat small intestinal G6PD were found to be 8.52 kcal/mol, 7.90 kcal/mol, 1.59, and 38 degrees C, respectively. The Km values for G6P and NADP+ were 70.1 +/- 20.8 and 23.2 +/- 7.6 microM, respectively. Double-reciprocal plots of 1/Vm versus 1/G6P (at constant [NADP+]) and of 1/Vm versus 1/NADP+ at constant [G6P]) intersected at the same point on the 1/Vm axis to give Vm = 53.8 U/mg protein.     

Subcellular Distribution Studies of Diadenosine Polyphosphates—Ap4A and Ap5A—in Bovine Adrenal Medulla: Presence in Chromaffin Granules

Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) have been identified in bovine adrenal medullary tissue using an HPLC method. The values obtained were 0.1 +/- 0.05 mumol/g of tissue for both compounds. The subcellular fraction where Ap4A and Ap5A were present in the highest concentration was chromaffin granules: 32 nmol/mg of protein for both compounds (approximately 6 mM intragranularly). This value was 30 times higher than in the cytosolic fraction. Enzymatic degradation of Ap4A and Ap5A, isolated from chromaffin granules, with phosphodiesterase produces AMP as the final product. The Ap4A and Ap5A obtained from this tissue were potent inhibitors of adenosine kinase. Their Ki values relative to adenosine were 0.3 and 2 microM for Ap4A and Ap5A, respectively. The cytosolic fraction also contains enzymatic activities that degrade Ap4A as well as Ap5A. These activities were measured by an HPLC method; the observed Km values were 10.5 +/- 0.5 and 13 +/- 1 microM for Ap4A and Ap5A, respectively.     

Trichomonas vaginalis: identification of a phospholipase A-dependent hemolytic activity in a vesicular subcellular fraction

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.     

Na+ for H+ exchange in rabbit erythrocytes

The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 microM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pH0 = 8.0, in the presence and absence of 60 microM 5,N,N-dimethyl-amiloride (DMA). The H+ efflux rate in a K+-containing medium was 116.38 +/- 4.5 mmol/l cell X hr. Substitution of Nao+ for Ko+ strongly stimulated H+ efflux to 177.89 +/- 7.9 mmol/l cell X hr. The transtimulation of H+ efflux by Nao+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 X 10(-7) M. The sequence of substrate selectivities for the external transport site were Na greater than greater than greater than Li greater than choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3-. The DMA sensitive H+ efflux was a saturable function of [Na+]o, with an apparent Km and Vmax of about 14.75 +/- 1.99 mM and 85.37 +/- 7.68 mmol/l cell X hr, respectively. However, the Nao+-dependent and DMA-sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 +/- 3.0 mmol/l cell X hr) which was abolished when pHo was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.     

H+-ATPase of crude homogenate of the outer mantle epithelium of Anodonta cygnea

The outer mantle epithelium of the freshwater bivalve, Anodonta cygnea, is responsible for the mineralisation of the shell. Under short circuit conditions, it generates a current that is due to the operation of an electrogenic proton pump located in the apical barrier of that epithelium. Bafilomycin A1 and Concanamycin A inhibited the short circuit current. The IC50 and maximum inhibition dose were 0.17 and 0.5 microM for Bafilomycin A1, and 0.7 and 5 microM for Concanamycin A. The present work was undertaken to further characterise V-type ATPase of the outer mantle cells. The V-ATPase enzymatic activity of crude homogenate, measured as the amount of inorganic phosphorous released, due to ATP hydrolysis, in the presence of Na2SO3 (200 mM) was found to be 4.6+/-1.1 micromol Pi/mg protein/h, at 27 degrees C, pH 7.0-7.4 and ATP 4.5-6.0 microM. Bafilomycin A1 and Concanamycin A inhibit the V-ATPase activity with an IC50 of 14 and 8 nmol mg(-1), respectively. Dicyclohexylcarbodiimide (DCCD; 100 mM) and NaNO3 (100 microM) inhibited the V-type ATPase in what it seems a non-specific manner and NEM (100 mM) was unable to do it. Bafilomycin A1 (10 microM) and Concanamycin A (10 microM), inhibited 50-60% of the total activity.     

Metabolism of opioid peptides by cerebral microvascular aminopeptidase M

Aminopeptidase M (EC 3.4.11.2), which can degrade low molecular weight opioid peptides, has been reported in both peripheral vasculature and in the CNS. Thus, we have studied the metabolism of opioid peptides by membrane-bound aminopeptidase M derived from cerebral microvessels of hog and rabbit. Both hog and rabbit microvessels were found to contain membrane-bound aminopeptidase M. At neutral pH, microvessels preferentially degraded low molecular weight opioid peptides by hydrolysis of the N-terminal Tyr1-Gly2 bond. Degradation was inhibited by amastatin (I50 = 0.2 microM) and bestatin (10 microM), but not by a number of other peptidase inhibitors including captopril and phosphoramidon. Rates of degradation were highest for the shorter peptides (Met5- and Leu5-enkephalin) whereas beta-endorphin was nearly completely resistant to N-terminal hydrolysis. Km values for the microvascular aminopeptidase also decreased significantly with increasing peptide length (Km = 91.3 +/- 4.9 and 28.9 +/- 3.5 microM for Met5-enkephalin and Met5-enkephalin-Arg6-Phe7, respectively). Peptides known to be present within or in close proximity to cerebral vessels (e.g., neurotensin and substance P) competitively inhibited enkephalin degradation (Ki = 20.4 +/- 2.5 and 7.9 +/- 1.6 microM, respectively). These data suggest that cerebral microvascular aminopeptidase M may play a role in vivo in modulating peptide-mediated local cerebral blood flow, and in preventing circulating enkephalins from crossing the blood-brain barrier.     

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.     

Protein kinase activities in neoplastic squamous epithelia and normal epithelia from the upper aero-digestive tract

In the present study the activities of three different protein kinase were determined in squamous cell carcinoma from the upper aero-digestive tract, and compared with the activities in normal oral mucosa. The protein kinases investigated are: a) cAMP-dependent protein kinase; b) cGMP-dependent protein kinase, and c) casein kinase II. The basal protein kinase activity, when histone IIa was used as substrate, was about 3-fold higher in tumors, as compared to normal mucosa, in the soluble fraction (32.0 +/- 4.2 and 10.9 +/- 2.4 pmol 32P/mg prot. X min, respectively). In the particulate fraction the basal protein kinase activity was about 9 times higher in tumors as compared to normal mucosa (19.4 +/- 5.2 and 2.1 +/- 0.3 pmol 32P/mg prot X min, respectively). The protein kinase activity in the presence of cyclic nucleotide (cAMP/cGMP) minus the basal protein kinase activity was taken as the cAMP- and the cGMP-dependent protein kinase activity, respectively. Maximal protein kinase activity was obtained in the presence of 0.5 microM of cyclic nucleotide both in squamous cell carcinoma and normal mucosa. In the cytosolic fraction the cAMP-dependent protein kinase activity was 33.9 +/- 13.0 pmol 32P/mg prot. X min in tumors, and 28.2 +/- 5.8 pmol 32P/mg prot. X min in normal tissue, after stimulation with 0.5 microM cAMP. The cGMP-dependent protein kinase activity was 5-10% of the cAMP-dependent protein kinase activity, and no concentration-dependent stimulation with cGMP was seen. The cGMP-dependent protein kinase activity in the presence of 0.5 microM cGMP was 2.4 +/- 1.3 and 1.8 +/- 0.6 pmol 32P/mg prot. X min in tumors and normal mucosa, respectively. Casein kinase II activity was determined only in the cytosolic fraction and was found to be 3-fold higher in tumors as compared to normal mucosa (31.8 +/- 5.2 and 8.6 +/- 3.5 pmol 32P/mg prot X min, respectively). This study shows a general increase in histone phosphorylation and casein kinase activity in neoplastic squamous epithelia compared to normal epithelia. No evidence for an increase in cyclic nucleotide dependent protein kinase activities in neoplastic squamous epithelia was found. This study thus supports the idea that phosphorylation/dephosphorylation reactions may play an important role in the control of cell growth, differentiation and proliferation.     

Intracellular calcium chelators and oxidant-induced renal proximal tubule cell death.

The effect of intracellular calcium chelators on rabbit renal proximal tubule (RPT) cell death induced by t-butyl hydroperoxide (TBHP) and H2O2 was examined. Preincubation of RPT suspensions with 50 microM QUIN 2/AM completely prevented TBHP (0.5 mM) and H2O2 (2 mM) induced cell death [i.e., release of lactate dehydrogenase (LDH)]. QUIN 2/AM, BAPTA/AM, EGTA/AM, and FURA 2/AM, at 5 microM, decreased LDH release (at 6 hr) from 41% to 4%, 21%, 26%, and 33%, and decreased lipid peroxidation (at 1 hr) from 1.0 to 0.1, 0.4, 0.6, and 0.8 nmol MDA/mg protein, respectively, after TBHP exposure. Since oxidant-induced lipid peroxidation and cell death are iron-dependent in this model, these results suggest that the intracellular calcium chelators inhibit cell death by chelating iron.     

Triglyceride lipase from the rabbit liver

Unlike the rabbit blood plasma the high-activity triglyceride lipase which does not require a protein cofactor and is resistant to 1 M of NaCl has been revealed in the liver of this animal. Rapid freezing of the liver tissue by liquid nitrogen before homogenization induced a sharp increase in the enzyme activity level and extraction of the active enzyme without heparin usually added to the homogenate. The specific activity of hepatic lipase purified from the ordinary heparin-containing homogenate by affinity chromatography on heparin-sepharose 4B was equal to 3800 microM of FFA/h per mg of protein. If the homogenate previously treated with liquid nitrogen was used as a source of enzyme, the specific activity of hepatic lipase was higher, namely, 13,000 and 19,000 microM of FFA/h per mg of protein in the presence or absence of heparin, respectively.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Mixed type inhibition of the renal Na+/H+ antiporter by Li+ and amiloride. Evidence for a modifier site

We studied the interactions of Na+, Li+, and amiloride on the Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pHin = 6.0; pHout = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (Km for Li+ = 1.2 +/- 0.1 mM; Km for Na+ = 14.3 +/- 0.8 mM; Vmax for Li+ = 2.40 +/- 0.07 fluorescence units/s/mg of protein; Vmax for Na+ = 7.10 +/- 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], [( Na+] = 90 mM; [Li+] less than 1 mM), we observed noncompetitive inhibition (Vmax for Na+/H+ exchange reduced by 25%). The apparent Ki for this noncompetitive inhibition was congruent to 50 microM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (Ki = 30 microM, Ki' = 90 microM) but was only a simple competitive inhibitor of Li+/H+ exchange (Ki = 10 microM). At [Li] = 1 mM and [amiloride] less than 100 microM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.     

Glucose transporters in isolated chromaffin cells. Effects of insulin and secretagogues.

1. Isolated chromaffin cells from bovine adrenal medulla were used to study glucose transport in a homogeneous neural tissue. 2. The affinity of glucose transporters was 1.20 +/- 0.52 mM by the infinite-cis technique and 1.02 +/- 0.09 mM by the direct transport experiments. 3. The affinity for 2-deoxyglucose of these transporters was 2.3 mM. 4. The glucose transporters, quantified by [3H]cytochalasin B binding, were 419,532 +/- 120,740 receptors/cell, which corresponds to about 7.2 +/- 2 pmol/mg of protein, with KD = 0.1 microM. 5. High-affinity insulin receptors with KD = 3.95 nM were present at a density of 68,400 +/- 7500 per cell. 6. Insulin and secretagogues increased glucose transport, raising the transporter number at the plasma membrane without changes in the affinity.