Evidence for membrane-bound carbonic anhydrase in the air bladder of bowfin (Amia calva), a primitive air-breathing fish.

The purpose of this study was to examine the subcellular distribution and isoenzyme characteristics of carbonic anhydrase from the gills and respiratory air bladder of bowfin Amia calva, a primitive air-breathing fish. Separation of subcellular fractions by differential centrifugation revealed that the vast majority of carbonic anhydrase from the gills of bowfin originated from the cytoplasmic fraction. Washing of the gill microsomal pellet also indicated that the carbonic anhydrase originally associated with this pellet was largely due to contamination from the cytoplasmic fraction. Experiments with a carbonic anhydrase inhibitor, sulphanilamide, and the plasma carbonic anhydrase inhibitor from this species confirmed that the bowfin gill probably contains only one carbonic anhydrase isoenzyme which had properties resembling those of CA II. In contrast to the situation in the gills, a relatively large percentage (27%) of the total air bladder carbonic anhydrase was associated with the microsomal fraction. Washing of the air bladder microsomal pellet removed little of the carbonic anhydrase activity, indicating that most of the carbonic anhydrase in the microsomal fraction was associated with the membranes. Like the mammalian pulmonary CA IV isoenzyme, microsomal carbonic anhydrase from the bowfin air bladder was less sensitive to the bowfin plasma carbonic anhydrase inhibitor, sodium dodecylsulphate (SDS) and sulphanilamide than was cytoplasmic carbonic anhydrase from the air bladder. Microsomal carbonic anhydrase from the bowfin air bladder also resembled CA IV in that it appears to be anchored to the membrane via a phosphatidylinositol-glycan linkage which could be cleaved by phosphatidylinositol-specific phospholipase C. Taken together, these results suggest that a membrane-bound carbonic anhydrase isoenzyme resembling mammalian CA IV in terms of inhibition characteristics and membrane attachment is present in the air-breathing organ of one of the most primitive air-breathing vertebrates.

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. I. Effect of Mg2+

Canine cerebellar membranes were fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). The effect of Mg2+ on inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release and [3H]IP3 binding was assessed. Mg2+ inhibited IP3-induced Ca2+ release in a concentration-dependent manner. Mg2+ influenced both the extent of IP3-induced Ca2+ release and the apparent affinity for IP3. A 10-fold change of free Mg2+ (from approximately 30 to approximately 300 microM) reduced the extent of Ca2+ release by two- to threefold and shifted the apparent Michaelis constant from approximately 0.5 to approximately 0.9 microM IP3. Thus Mg2+ seemed to be noncompetitive inhibitor of IP3-induced Ca2+ release. Mg2+ also inhibited Ca2+ release elicited by glycerophosphoinositol 4,5-bisphosphate, a poorly metabolized analogue of IP3. Mg2+ and heparin sodium were shown to be additive inhibitors of IP3-induced Ca2+ release. Mg2+ inhibited [3H]IP3 binding under experimental conditions designed to minimize IP3 hydrolysis. Scatchard plots indicated that 0.5 mM free Mg2+ reduced maximum binding from 10.9 to 3.5 pmol IP3 bound/mg protein and increased the dissociation constant from 136 to 227 nM. The modulation of [3H]IP3 binding and IP3-induced Ca2+ release by Mg2+ could be physiologically relevant.

Intraenterocyte distribution of absorbed lipid and effects of phosphatidylcholine

The present studies were designed to investigate the distribution of absorbed lipid in intestinal mucosal cells and to identify the chylomicron precursor pool. Rats were infused intraduodenally with glyceryl tri[9,10(n)-3H]oleate (135 mumol/h); other rats were in addition infused with 9 mumol/h of phosphatidylcholine. After 5-h infusion the proximal one-half of intestine was removed and the mucosa obtained. It was found that 50% of the radioactivity in the whole homogenate pelleted on centrifugation at 75,000 g.min. The supernatant was further fractionated by high-speed centrifugation resulting in a floating lipid layer, a supernatant, and a microsomal pellet. The results showed that these subcellular fractions had a triacylglycerol specific activity 46-52% of the infusate's specific activity. Including phosphatidylcholine in the duodenal lipid infusion increased the triacylglycerol specific activity of all subcellular fractions (70%) resulting in a specific activity approaching that of the infusate, which would be expected of chylomicron triacylglycerol. These studies demonstrate 1) that considerable mucosal lipid is distributed into a low-speed pellet, 2) that mucosal triacylglycerol specific activity can be greatly increased by including phosphatidylcholine in a lipid infusion, and 3) that despite obtaining multiple subcellular fractions, the chylomicron precursor pool could not be clearly identified in the mucosa of control rats.

Characterization of a strain-specific inhibitor of cell division from Amoeba cytoplasm

The microinjection of cytoplasm taken from one strain of large free-living amoeba into another strain is followed by an incompatibility phenomenon, the inhibition of division amongst the recipient cells. The post-microsomal supernatant fraction from Amoeba discoides (T1D13) injected into A. proteus (T1P) inhibited division in 90% of the injected cells. Further centrifugation of this fraction yielded a pellet which when resuspended and injected, inhibited division in over 95% (and sometimes 100%) of the cells. No inhibitory activity remained in the supernatant after the removal of this pellet. Treatment with 10 micrograms/ml trypsin destroyed the activity of this pellet, while 25 micrograms/ml ribonuclease reduced the inhibitory activity by approximately 40%. Passage of the resuspended post-microsomal pellet through Sephadex G-200 gave one main peak of material which eluted in the void volume. Concentration of this material by either dialysis or lyophilization followed by microinjection into A. proteus showed that this void volume peak contained the inhibitory material, although the most active preparations did not give more than 66% inhibition of division. After elution from Sephadex, the void volume material was analysed by electrophoresis under non-denaturing and denaturing conditions, and by isoelectric focusing. One problem was the loss of inhibitory activity after keeping the pellet at 4 degrees C for 4–5 days, which made further analysis by microinjection difficult. Preliminary experiments using a post-microsomal pellet prepared from Dawson's A. proteus (DP) which inhibited division in A. proteus (T1P) gave a similar profile after Sephadex chromatography and gel electrophoresis.

Chromogenic Assays for the Determination of Prothrombin Related Material in Rat Liver Fractions.

Clotting assays are not easily applied to turbid solutions such as microsomal fractions. With the development of chromogenic substrates, the esterolytic activity of prothrombin related material can be assayed biochemically in such systems. Liver fractions were prepared by differential centrifugaron. Liver homogenate was centrifuged at 10,000 g. for 10 minutes to yield supernatant 1.Supernatant 1 was further centrifuged at 105,000 g for 60 minutes to yield the microsomal pellet and supernatant 2. Taipan and Echis carinatus snake vanoms were used to generate esterolytic activity in the various liver fractions. In all fractions the esterolytic activity generated by E. carinatus venom was greater than that generated by Taipan Venom. Both assays indicated that the microsomal pellet had similar esterolytic activity to supernatant 2. When liver fractions were prepared from warfarin treated rats, the assays revealed that the relative proportions of prothrombin related material in the fractions had altered. The esterolytic activity of the microsomal fraction was found to be greatly increased whilst supernatant 2 had no detectable activity.Warfarin treated rats have greatly decreased levels of prothrombin in the plasma due to inhibition of vitamin K-dependent carboxylation in the liver. It is suggested that the lack of prothrombin in the plasma reflects the lack of soluble prothrombin in supernatant 2, and the concomitant build-up of precursor forms bound to the microsomes.

The purification and specificity of a neutral endopeptidase from rabbit kidney brush border

1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [125I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [125I]iodoglucagon at a rate comparable with that for [125I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the α-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.

Metabolism of androst-4-ene-3,17-dione by subcellular fractions of rat adrenal tissue with particular reference to microsomal C19-steroid 5α-reductase

The location and some characteristics of rat adrenal C19-steroid 5α-reductase were investigated by using [7α-3H]androst-4-ene-3,17-dione and [7α-3H]testosterone as substrates. The enzymes system was shown to be NADPH-dependent and associated with the microsomal fraction. In addition, some evidence was also obtained for the existence of a separate NADH-dependent system in the soluble fraction. Further investigation of androst-4-ene-3,17-dione metabolism by subcellular fractions indicated the presence of NADH-dependent 3α- and 3β-hydroxy steroid dehydrogenase systems in the microsomal pellet. This pellet also appeared to contain an NADH-dependent 17β-hydroxy steroid dehydrogenase system, and a similar though separate system was detected in the cytosol. Malate (20mm) effectively inhibited the microsomal C19-steroid 5α-reductase, which showed similar values for Km and Vmax. when either androst-4-ene-3,17-dione or testosterone was used as substrate. Cytochrome c was added to all incubation mixtures used for the determination of these values to inhibit the formation of metabolites other than 5α-androstane-3,17-dione and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) respectively. It was also found that corticosterone did not inhibit the 5α-reduction of androst-4-ene-3,17-dione under these conditions, indicating that separate enzymes exist for the 5α-reduction of C19- and C21-steroids in the rat adrenal.

Factors affecting protein synthesis in rat kidney ribosome preparations

Kidney ribosomes that were prepared from the postmitochondrial supernatant or the microsomal pellet of homogenates by deoxycholate treatment consisted mainly of monomers and dimers rather than polysomes. The addition of liver postmicrosomal supernatant to kidney homogenates or to kidney microsomes did not increase the polysome yield or the incorporation of 14C-leucine into hot acid-insoluble protein. In contrast to kidney, liver ribosomes that were prepared in the presence of liver supernatant (containing ribonuclease inhibitor) had an increased proportion of polysomes and an increased incorporation of 14C-leucine into protein compared to liver ribosomes prepared in the absence of supernatant.The incorporation of 14C-leucine into protein in kidney ribosome preparations was considerably lower than it was in liver ribosome preparations. The incorporation depended on the concentration of Mg2+, GTP, and pH 5 enzymes. Following preincubation of kidney ribosomes to eliminate endogenous mRNA, the incorporation of 14C-phenylalanine into peptide using either phenylalanine or phenylalanyl-tRNA was found to depend on the addition of poly U.Ribonuclease activity in kidney supernatant and pH 5 enzyme preparations was much greater than it was in liver preparations, and may partly account for the lower polysome yield and protein synthesis in kidney.

Studies on the biological control of glycogen metabolism in liver. III. Subcellular distribution of glycogen metabolizing enzymes

In a study of the subcellular distribution of the enzymes of the glycogen cycle in rat liver, glycogen synthetase was found in both the microsomal pellet and supernatant fractions, but the activity was always greater in the pellet than in the supernatant fraction. Variations in the concentration of liver glycogen, brought about by fasting and refeeding, did not alter this distribution. In agreement with the results of others, phosphorylase appeared almost entirely in the microsomal fraction, presumably bound to glycogen, when liver glycogen was high, or in the supernatant fraction when liver glycogen was low. These results indicate that synthetase and phosphorylase may be bound with different affinities or to different forms of glycogen.Regardless of whether the animals were fasted or fed, significant amounts of UDPG pyrophosphorylase activity appeared in the microsomal pellet fraction. The remainder of UDPG pyrophosphorylase and all of phosphoglucomutase activities were present in the microsomal supernatant fraction. As synthetase and phosphorylase are bound to glycogen, which in turn is known to be associated with the endoplasmic reticulum, the occurrence of UDPG pyrophosphorylase, synthetase, and phosphorylase in the microsomal fraction may have functional significance to the glycogen cycle.

The biological conversion of 7-dehydrocholesterol into cholesterol and comments on the reduction of double bonds

It is shown that the 7-dehydrocholesterol reductase-catalysed conversion of 7-dehydrocholesterol into cholesterol (II), with a 105000g microsomal pellet of rat liver in the presence of [4−3H2]NADPH, results in the transfer of radioactivity to the 7α-position of cholesterol. When the conversion is carried out in the presence of tritiated water the label is introduced exclusively at the 8β-position. However, when the conversion of 7-dehydrocholesterol into cholesterol is performed with a 500g supernatant of rat liver homogenate the radioactivity is incorporated at both the 7α- and the 8β-position. Evidence is provided for the presence of an enzyme system in the 500g supernatant that catalyses an equilibration of hydrogen atoms between those at the 4-position of NADPH and those of water. The work with stereospecifically labelled cofactors shows that both the equilibrating system and the 7-dehydrocholesterol reductase utilize the 4B-hydrogen atom of NADPH. In the light of these results a mechanism for the reduction of carbon–carbon double bonds is discussed.