Prenatal induction of tyrosine aminotransferase in rat liver

• 1.1. Hepatic tyrosine aminotransferase activity from adult rat can be resolved into four components on hydroxylapatite column.
• 2.2. A similar profile of enzyme distribution can be obtained from late foetal liver.
• 3.3. Insulin administration to pregnant rats result in induction of two isoenzymes of tyrosine aminotransferase in foetal rat liver. Similarly Cyclic AMP injection to foetal rats in utero results in the induction of the same two forms of the enzyme.
• 4.4. Triaminolone injection to foetal rats in utero leads to the induction of three of the isoenzymes of tyrosine aminotransferase.

     

Preparation of two neurotoxic esterases from the chick central nervous system

• 1.1. A standard procedure for lipid-extraction of lyophilized hen brain material is decribed.
• 2.2. Nine carboxylesterase isoenzymes (EC 3.1.1.1) are identified in lipid-extracted lyophilized material (LELM) using kinetic analysis of organophosphate inhibition. Total phenyl valerate (PV) hydrolysing carboxylesterase activity in LELM is 43.3U.g⁻¹
• 3.3. Two carboxylesterase isoenzymes of LELM are classified as neurotoxic esterases (NTE_A and NTEg_B).
• 4.4. Using n-octylglucoside 51% of the water-insoluble neurotoxic esterase activity from LELM are solubilized.
• 5.5. Six carboxylesterase isoenzymes including NTE_A (6.5 U-l⁻¹) and NTE_B (4.2 U-l⁻¹) are present in the solubilized preparation.
• 6.6. Throughout purification and separation steps carboxylesterase isoenzymes are identified by their rate constants for the reaction with organophosphorus inhibitors.

     

Ostrich α-amylase: Purification and characterization of the pancreatic isoenzymes

• 1.1. Four ostrich pancreatic α-amylase isoenzymes were isolated by isoelectric focusing, following affinity chromatography on cyclohepta-amylose-Sepharose 4B.
• 2.2. Amino acid compositions of the four isoenzymes are very similar with only one charged amino acid (Arg) being significantly different.
• 3.3. The molecular weights, as determined by SDS-PAGE and amino acid composition, are nearly identical (52–53 kDa) for all four isoenzymes.
• 4.4. The four α-amylase isoenzymes appear to be kinetically distinct enzymes with a requirement for calcium.
• 5.5. Ostrich α-amylase isoenzymes appear to be non-glycosylated and contain one free thiol group.

     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

     

Purification and partial characterization of the alkaline phosphatase from Allolobophora caliginosa

• 1.1. A purification of the enzyme from the starting material was achieved by means of butanol and acetone fractionations and, successively, by DEAE cellulose and Sephadex G-200 chromatographies.
• 2.2. Two enzymatic forms were separated; they showed various similar characteristics but differed greatly in specific activity.
• 3.3. It is probable that in A. caliginosa a sole alkaline phosphatase form exists and the less active fraction is partly denatured enzyme.
• 4.4. It is not completely possible to exclude the existence of two isoenzymes.

     

Subunit homologies of lactate dehydrogenase (LDH) isoenzymes between amphibians (Xenopus laevis) and mammals (Wistar rat)

• 1.1. The subunit distribution and subunit homologies of LDH isoenzymes were studied in the amphibian Xenopus laevis and in Wistar rats.
• 2.2. Several of the 11–15 isoenzymes of the pattern in Xenopus, separable by vertical starch gel electrophoresis, were purified, hybridized, and the cross-reaction of antibodies against the most positively charged isoenzyme with the isoenzymes present in tissue extracts of both species was tested.
• 3.3. The isoenzyme with the highest positive charge in Xenopus is a M-homotetramer homologous to mammalian LDH₅.
• 4.4. The multibanded pattern of Xenopus LDH isoenzymes is very probably due to heterozygoty of the gene locus controlling the synthesis of M-subunits rather than to an epigenetic subbanding phenomenon.

     

Red cell carbonic anhydrase levels in flounders,Platichthys flesus L., from salt water and fresh water

• 1.1. Carbonic anhydrase levels in flounder red cells were unchanged by adaptation to salt or fresh water.
• 2.2. Two major red cell isoenzymes were found in flounder red cells after electrophoresis. These patterns were identical in all fish studied, whether from salt or fresh water environments. Thus no inherited or adaptive changes were observed.
• 3.3. Both flounder red cell carbonic anhydrase isoenzymes split a range of ester substrates. Activity was abolished with acetazolamide.

     

Hormonal regulation of human apolipoprotein E gene expression in HepG2 cells

• 1.1. Hormonal regulation of apolipoprotein E (apoE) gene expression by insulin and thyroid hormone was studied in a human hepatoma cell line, HepG2.
• 2.2. Changes at the mRNA level, mRNA translation, in vivo synthesis and secretion were monitored.
• 3.3. Both insulin and triiodothyronine were found to have no significant effect on apoE mRNA levels.
• 4.4. Insulin treatment caused an inhibition of: (a) the in vitro translation of endogenous apoE mRNA in a HepG2 cell-free system (25%), and (b) the incorporation of radioactivity into newly-synthesized apoE in an in vivo pulse-chase labeling experiment (32%).
• 5.5. Interestingly, apoE secretion rate was found to be significantly reduced with insulin (84%) suggesting that a major portion of newly-synthesized apoE may be shunted into a degradative pathway.
• 6.6. Using a similar experimental approach, triiodothyronine showed no significant effect on the rate of apoE synthesis or translation (6–15% decrease), however a slight reduction (20%) in secretion rate was shown.
• 7.7. Overall, apoE gene expression does not appear to be influenced by triiodothyronine significantly but is modulated by insulin at the translational and post-translational level.

     

Changes in malate dehydrogenase isoenzymes during differentiation of rabbit bone marrow erythroid cells

• 1.l. Malate dehydrogenase activity was assayed in rabbit bone marrow erythroid cells which had been separated by velocity sedimentation into six fractions corresponding to different stages of development.
• 2.2. During differentiation enzyme activity decreased 10-fold and one of the two isoenzymes present in immature cells was lost.
• 3.3. The most extensive change occurred in orthochromatic erythroblasts immediately after condensation of the nucleus.

     

Purification and characterization of camel liver glutathione S-transferase

• 1.1. Glutathione S-transferases have been purified (18-fold) in 65–70% yield from the liver of one humped camel using affinity chromatography on glutathione-linked agarose.
• 2.2. Chromatofocusing technique resolves the glutathione S-transferases into seven distinct isoenzymes with apparent pI of 8.7, 8.4, 8.0, 7.8, 7.3 and 6.5.
• 3.3. The major isoenzyme (pI 8.7) which accounted for over 95% of the total activity was composed of two identical subunits of molecular mass 24,000 and was immunologically similar to the other six isoenzymes.
• 4.4. The substrate specificities and the effect of various inhibitors on the activity of the abundant camel liver isoenzyme were also examined.

     

Effect of isolation methods and of possible genetic factors on the multiple isoenzymic forms of pig muscle phosphoglucose isomerase

• 1.1. Pig muscle phosphoglucose isomerase has been found to exist in three isoenzymic forms.
• 2.2. Procedural artifacts, such as fractionation and chromatography methods, inadvertent oxidation during preparative work-up and post-mortem aging effects prior to commencement of isolation from the muscle tissue, have been eliminated as causes for the presence of the multiple enzyme forms.
• 3.3. Studies on the amino acid and peptide compositions of the individual isoenzymes yielded small, but distinct differences in primary structure.
• 4.4. These findings, in conjunction with literature reports on pig breeding studies, are interpreted to suggest that genetic determinants are the primary origin of the isoenzymes with secondary, possibly nongenetic, factors being responsible for the relative distributions of the three enzyme forms.

     

One and two dimensional isoelectric focusing of lactate dehydrogenase from the tissues of Ovis aries

• 1.1. Lactate dehydrogenase in ovine tissues was separated by electrophoresis, one dimensional isoelectric focusing (IEF) and a two dimensional technique.
• 2.2. Tissues showed five zones of enzyme activity consisting of multiple bands after IEF.
• 3.3. The IEF zymograms were unique for each tissue and differed from those of other species.
• 4.4. Two dimensional separation revealed that the five zones of activity observed on IEF corresponded to the five isoenzymes separable by conventional electrophoresis.

     

Rat liver endoplasmic reticulum protein kinases

• 1.1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography.
• 2.2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase and casein kinases.
• 3.3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis.
• 4.4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca²⁺ /calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

     

The influence of ehrlich ascites tumour growth on the turnover of lactate dehydrogenase in tissues of the mouse

• 1.1. The influence of Ehrlich ascites tumour growth on the turnover of total soluble protein and lactate dehydrogenase (LDH) in mouse tissues has been studied.
• 2.2. Turnover parameters were determined by means of double-labelling technique, with the enzyme (LDH) being isolated by affinity chromatography.
• 3.3. Tumour growth was accompanied by a decreased rate of synthesis of total protein in all tissues.
• 4.4. Lactate dehydrogenase by contrast snowed an increased rate of synthesis in all tissues but kidney.
• 5.5. These directions of change, in combination with the lesser response of degradation constants, resulted in a consequent conservation of enzyme activity in all tissues except kidney.
• 6.6. A generalized shift in the LDH isozyme pattern of these tissues was also observed during tumour growth with an increased contribution of A-type subunit.
• 7.7. These results have been discussed in relation to the redirection of protein synthesis and degradation, the occurrence of foetal isozymes, and possible mechanisms involved in the redistribution of protein resources in the animal during tumour development.

     

Malate dehydrogenase and non-specific esterase isoenzymes of eggs of the honey bee (Apis mellifera L.)

• 1.1. Isoelectric focusing on polyacrylamide gels was used to detect malate dehydrogenase and non-specific esterase isoenzymes in diploid honey bee (Apis mellifera L.) eggs of several known ages.
• 2.2. It was determined that the number of MDH and EST isoenzymes increased as development proceeded with an apparent increase in the quantity of some of the isoenzymes.

     

Serum amylase in the african elephant, (Loxodonta africana)

• 1.1. The α-amylase activity in the serum of the African elephant has been measured by 2 different methods. Consistently high values were obtained, about 10 times those of normal human serum measured by the same methods.
• 2.2. Electrophoretic separation of amylase isoenzymes revealed a single band in the γ-globulin region and a group of 4 bands in the β-globulin region.
• 3.3. Both sets of findings are discussed in relation to results reported on other mammals.

     

Isolation and characterization of alpha-foetoprotein from foetal pigs

• 1.1. Porcine α-foetoprotein, APP, was isolated from serum of pig foetuses using preparative electrophoresis and irnmunosorbent techniques.
• 2.2. The purity of the isolated AFP was controlled by polyacrylamide gel electrophoresis, electro-immunoassay and by injection of isolated AFP into rabbits.
• 3.3. During the preparation of AFP from foetal pig serum an unexpected α₁-globulin appeared as a contaminating protein.
• 4.4. The molecular weight, isoelectric point and sedimentation coefficient were found to be 70,000, 4–61 and 4–56 respectively. No microheterogeneity as regards the physicochemical parameters investigated could be seen. The results were compared with those reported for some other mammals.

     

Evidence that the 5' end of rabbit globin mRNA is hybridized with its 3' end

• 1.1. A radiopolyadenylated rabbit globin mRNA was treated with different concentrations of ribonuclease V₁ from cobra venom.
• 2.2. The enzymatic digests were chromatographed on an aminophenylboronate-agarose column, which specifically captured the cap structure i.e. n⁷G(5') ppp (5') NmP.
• 3.3. When the capture fragment was chromatographed on a Sephadex G-100 column, its size was smaller than the native molecule and also bore radioactivity, i.e. a poly(A) tail.
• 4.4. These results provide evidence that the 5' end (which encompasses the cap structure) of rabbit globin mRNA is hybridized and in close proximity to its 3' end.
• 5.5. We conclude that this conformation is required for messenger translation efficiency.