Heterogeneity of expression and secretion of native and mutant [AspB10]insulin in AtT20 cells

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.     

Experiments on the central pattern generator for swimming in amphibian embryos

The central nervous system of paralysed Xenopus laevis embryos can generate a motor output pattern suitable for swimming locomotion. By recording motor root activity in paralysed embryos with transected nervous systems we have shown that: (a) the spinal cord is capable of swimming pattern generation; (b) swimming pattern generator capability in the hindbrain and spinal cord is distributed; (c) caudal hindbrain is necessary for sustained swimming output after discrete stimulation. By recording similarly from embryos whose central nervous system was divided longitudinally into left and right sides, we have shown that: (a) each side can generate rhythmic motor output with cycle periods like those in swimming; (b) during this activity cycle period increases within an episode, and there is the usual rostrocaudal delay found in swimming; (c) this activity is influenced by sensory stimuli in the same way as swimming activity; (d) normal phase coupling of the left and right sides can be established by the ventral commissure in the spinal cord. We conclude that interactions between the antagonistic (left and right) motor systems are not necessary for swimming rhythm generation and present a model for swimming pattern generation where autonomous rhythm generators on each side of the nervous system drive the motoneurons. Alternation is achieved by reciprocal inhibition, and activity is initiated and maintained by tonic excitation from the hindbrain.     

Metabolism of L-beta-lysine by a Pseudomonas. Purification and properties of a deacetylase-thiolesterase utilizing 4-acetamidobutyryl CoA and related compounds

A deacetylase-thiolesterase that cleaves both the amide and thiolester bonds of 4-acetamidobutyryl CoA has been highly purified from extracts of Pseudomonas B4 grown in a medium containing L-beta-lysine (3,6-diaminohexanoate) as the main energy source. The enzyme has a molecular weight of about 275,000 and contains 8 apparently identical subunits of 36,500 daltons. Products of 4-acetamidobutyryl CoA degradation are stoichiometric amounts of CoASH and acetate, variable amounts of 4-aminobutyrate and its lactam, 2-pyrrolidinone, and a little 4-acetamidobutyrate. The relative yields of 4-aminobutyrate and 2-pyrrolidinone are determined by the enzyme level. At high enzyme levels the 4-aminobutyrate/pyrrolidinone ratio is about 2, whereas at low enzyme levels only pyrrolidinone is formed. Under the latter conditions, 4-aminobutyryl CoA accumulates transiently and is converted nonenzymatically to pyrrolidinone and CoASH. Since the enzyme does not form 4-aminobutyrate from synthetic or enzymatically formed 4-aminobutyryl CoA, we conclude that a 4-aminobutyryl CoA-enzyme complex is the actual precursor of 4-aminobutyrate, whereas free 4-aminobutyryl CoA is the precursor of pyrrolidinone. Several analogs of 4-acetamidobutyryl CoA containing different amino acid or amide moieties, and several simple acyl CoA compounds are utilized by the enzyme; 4-propionamidobutyryl CoA and 5-acetamidovaleryl CoA are most readily decomposed. Acetyl CoA is a very poor substrate. 3-Acetamidopropionyl CoA is first converted to acetate and beta-alanyl CoA and the latter compound is slowly hydrolyzed to beta-alanine and CoASH. Little deacetylase-thiolesterase is formed by bacteria grown in absence of beta-lysine, but another thiolesterase, lacking deacetylase activity, is produced. The deacetylase-thiolesterase catalyzes an essential step in the aerobic degradation of L-beta-lysine.     

Enzymes involved in 3,5-diaminohexanoate degradation by Brevibacterium sp.

Cell-free extracts of Brevibacterium sp. L5 grown on DL-erythro-3,5-diaminohexanoate were found to contain a 3-keto-5-aminohexanoate cleavage enzyme that converts 3-keto-5-aminohexanoate and acetyl-coenzyme A (CokA) to 3-aminobutyryl-CoA and acetoacetate and a deaminase that coverts L-3-aminobutyryl-CoA to crotonyl-CoA. The cleavage enzyme has been purified extensively, and some of its properties have been determined for comparison with the 3-keto-6-acetamido-hexanoate cleavage enzyme of Pseudomonas sp. B4. The deaminase has been partially purified and characterized. Both the cleavage enzyme and the deaminase are induced by growth on 3,5-diaminohexanoate. The presence of these and other accessory enzymes in Brevibacterium sp. extracts accounts for the results of earlier tracer experiments which showed that C-1 and C-2 of 3-keto-5-aminohexanoate are converted mainly to acetoacetate and acetate, whereas C-3 to C-6 are converted mainly to 3-hydroxybutyrate or its coenzyme A thiolester. The enzymes observed in extracts of Brevibacterium sp. can account for the conversion of 3,5-diaminohexanoate to acetyl-CoA.     

Isozyme shift in cultured fetal human hepatocytes: a study of pyruvate kinase and phosphofructokinase

Hepatocytes from a 4-month old fetus were cultured for 15 days. We found that fetal hepatocytes contained some R₁ (precursor) form of L-type pyruvate kinase. Culture was associated with a considerable increase of the M₂-type pyruvate kinase activity, but some L-type enzyme could be detected even after 10 days.Isozyme shift of phosphofructokinase seemed to be a progressive rather low phenomenon. Fetal hepatocytes showed an increase of the F-type form and a disappearance of the M-type form during culture. However, by day 10, the L-type enzyme remained predominant; this is in striking contrast with the findings reported on cultured fibroblasts.From these results, pyruvate kinase can be considered as a “strong” marker of cell differentiation, while phosphofructokinase is rather a “weak” marker.     

Purification and partial characterization of different forms of phosphofructokinase in man.

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from human muscle, brain, heart and granulocytes has been purified using a two or three step purification procedure. The main step is Blue Dextran-Sepharose 4B chromatography with selective elution of phosphofructokinase by formation of the ternary complex ADP or ATP-fructose-6-P-enzyme. Muscle and heart contain only enzyme subunits with a molecular weight of 85,000. This type of subunit is predominnant in brain, where it co-exists with subunits of about 80,000 daltons. A single type of subunits is found in the granulocytes, with a molecular weight of 80,000. Anti-muscle phosphofructokinase antiserum reacts only with M-type enzyme. Anti-granulocyte enzyme antiserum, absorbed by pure brain phosphofructokinase, exhibits a narrow specificity against the so-called L-type enzyme. Anti-brain antiserum, absorbed by pure muscle phosphofructokinase and partly purified red cell enzyme, exhibits a narrow specificity against a phosphofructokinase form predominant in fibroblasts and present in brain (F-type).