基因工程人胰岛素原和胰岛素的分离纯化及性质研究

E. coli DH 5 alpha cells harboring a plasmid pWR 590-BCA 4 for fused human proinsulin production were cultured. The fused human proinsulin was isolated from the fermented cells and then subjected it to cleavage with BrCN. The cleaved product was then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis. The isolation of human proinsulin-S-sulfonate was accomplished by ion exchange chromatography on QAE-sephadex A-25, followed by gel filtration on sephadex G-50. The purified human proinsulin-S-sulfonate was folded using a disulfide interchange method. The folding mixture was then chromatographed on sephadex G-50 and purified proinsulin was obtained. The proinsulin was then converted to human insulin and C-peptide by a combination cleavage with trypsin and carboxypeptidase B. The total yield of human insulin was about 5 mg/L The Zinc insulin crystals were obtained with amorphous human insulin using citrate method. The amino acid composition N-terminal sequences as well as C-terminal amino acid residues are in agreement with expected results. The hypoglycemic activity of purified human insulin is 26-27 U/mg, as judged by mouse convulsion assay, and the RIA activity is about 99% of that of porcine insulin.     

Isolation and characterization of cells from rat adipose tissue developing into adipocytes.

To identify cells developing into adipocytes by accumulation of triglyceride, rat epididymal fat pad cells from small rats were exposed to (3)H-labeled chylomicron fatty acids in vivo and then liberated with collagenase. Tissue remnants were removed by filtration and mature fat cells by flotation. Aggregating cells were then removed by filtration through a 25- micro m nylon screen. Further purification of cells labeled in vivo was obtained by removing floating cells from those adhering to the bottom of a culture dish. The adhering cells multiplied to a confluent monolayer when cultured in Medium 199 containing serum, glucose, insulin, and a triglyceride emulsion. The cells then gradually enlarged due to granulation of the cytoplasm by a lipid-staining material. After about 2 weeks these granules had coalesced forming mature adipocytes of typical signet-ring appearance. Free adipocytes could then be recovered from the cultures by collagenase treatment. After about 2 weeks of culture these cells had the same size (about 30 micro m) as adipocytes recovered in the original collagenase preparation of the rat epididymal fat pad. They contained triglyceride lipase activity and incorporated glucose into triglycerides to the same extent as cells developed in vivo but had higher lipoprotein lipase activity. In vitro, heparin in a low concentration, prostaglandin E(1), isobutylmethylxanthine, and cholera toxin markedly promoted the development of these cells into adipocytes. This could be shown to occur almost completely indicating that this fraction of cells was homogeneous and consisted of cells with the capacity to form adipocytes. The duplication time was about 2 days and did not change with subculturing. Preadipocytes could be obtained by density gradient centrifugation, isolating triglyceride-containing cells either directly from the pad or after 3 days in culture. All of these cells developed into adipocytes as described above but did not multiply as readily. It was concluded that cells from the epididymal fat pad from small rats can be isolated in a homogenous fraction that develops in culture into cells of identical morphology and function as adipocytes formed in vivo. The differentiation of these cells into adipocytes may be manipulated in vitro.     

Semisynthetic human [[3H2]Phe1]proinsulin.

Biosynthetic human proinsulin (obtained by recombinant DNA techniques) was used as the starting material for the preparation, by semisynthetic methods, of [3H]proinsulin with the label at the N-terminal phenylalanine residue. The labelled proinsulin was characterized by its retention time on reversed-phase h.p.l.c., by polyacrylamide-gel electrophoresis, by the time course of its enzymic conversion into insulin and by chromatographic analysis after extensive proteolytic degradation. The specific radioactivity of the product was 5 Ci/mmol. Experimental details of the preparation of human [[3H]Phe1]proinsulin, the isolation of this product by isocratic h.p.l.c. and gel filtration, and further characterization of protein intermediates have been deposited as supplement SUP 50138 (12 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on prepayment [see Biochem. J. (1987) 241, 5].     

Biological properties of antibodies against rat adipocyte intrinsic membrane proteins. Dependence on multivalency for insulin-like activity.

Antisera from rabbits injected with rat adipocyte plasma membranes or intrinsic proteins from such membranes, obtained by a dimethylmaleic anhydride extraction step, mimicked the action of insulin on both glucose transport and lipolysis in intact adipocytes. Biological activity in both types of antisera was mediated by immunoglobulin binding to one or more intrinsic proteins of the adipocyte plasma membrane since fat cells were unresponsive to all antisera absorbed with dimethylmaleic anhydride-extracted membranes. Acid treatment of immunoprecipitates released antibodies which activated glucose uptake and reacted with solubilized adipocyte membranes on immunodiffusion plates. The biologically active immunoglobulin preparations failed to form immunoprecipitin lines when tested against membranes from brain, liver, lung, muscle, kidney, and spleen. Insulin-sensitive glucose uptake in rat soleus muscle did not respond to the antisera. The antibodies activated hexose uptake into fat cells and reacted with solubilized adipocyte membranes on immunodiffusion plates when rat or mouse adipocytes were studied, but not when monkey fat cells were used. The anti-membrane antibody preparations readily activated hexose uptake in trypsinized fat cells which had lost the capacity to bind or respond to insulin. These data are consistent with the concept previously proposed (Pillion, D.J., and Czech, M.P. (1978) J. Biol. Chem. 253, 3761-3764) that the anti-membrane immunoglobulins do not interact with the insulin binding site of the insulin receptor. Monovalent Fab fragments of the biologically active antisera, prepared by papain digestion of the native anti-membrane immunoglobulins, were ineffective in enhancing glucose uptake in adipocytes. However, biological activity of the anti-membrane Fab fragments was restored by the addition of goat anti-rabbit Fab antisera to cells treated with the Fab fraction. Anti-rabbit Fab antisera alone or in combination with Fab fragments prepared from control rabbit sera exhibited no biological activity. These results demonstrate that the ability of anti-membrane antisera to mimic the biological activity of insulin on isolated fat cells is critically dependent on immunoglobulin binding to one or more intrinsic plasma membrane proteins and the multivalent nature of immunoglobulin structure.     

Studies of the biological activity of insulin, cyclic nucleotides and concanavalin A in the isolated fat cell.

The insulin or proinsulin response of the isolated rat adipocyte was destroyed by preincubation with trypsin. After 120 minutes, biological responsiveness partially regenerated. Similarly, the biological responsiveness of the isolated fat cell to non-suppressible insulin-like activity (NSILA) was only partially destroyed following trypsin digestion, and did not regenerate. In contrast to the above, cyclic AMP or dibutyryl cyclic AMP effects were unaltered by trypsin or neuraminidase digestion.     

The effect of placement of tryptophan residues in selected A-chain positions on the biological profile of insulin

In continuation of our efforts to study the solution structure and conformational dynamics of insulin by time-resolved fluorescence spectroscopy, we have synthesized and examined the biological activity of five insulin analogues in which selected naturally occurring residues in the A-chain have been replaced with the strongly fluorescent tryptophan residue. The potency of these analogues was evaluated in lipogenesis assays in isolated rat adipocytes, in receptor binding assays using rat liver plasma membranes, and in two cases, in receptor binding assays using adipocytes. [A3 Trp]insulin displays a potency of 3% in receptor binding assays in both liver membranes and in adipocytes, but only 0.06% in lipogenesis assays as compared to porcine insulin. [A10 Trp] insulin displays a potency ofca. 40% andca. 25% in rat liver receptor binding and lipogenesis assays, respectively. [A13 Trp]insulin displays a potency ofca. 39% in rat liver receptor binding assays, but onlyca. 9% in receptor binding in adipocytes; in lipogenesis assays, [A13 Trp] insulin displays a potency ofca. 12%, comparable to its potency in adipocyte receptor binding assays. [A15 Trp]insulin exhibits a potency of 18% and 9% in rat liver receptor binding and lipogenesis assays, respectively. The doubly substituted analogue, [A14 Trp, A19 Trp] insulin, displays a potency ofca. 0.7% in both rat liver receptor binding assays and lipogenesis assays. These data suggest two major conclusions: (1) the A3 and A15 residues lie in sensitive regions in the insulin molecule, and structural modifications at these positions have deleterious effects on biological activity of the hormone; and (2) [A13 Trp]insulin appears to be a unique case in which an insulin analogue exhibits a higher potency when assayed in liver tissue than when assayed in fat cells.     

Receptor-mediated degradation by rat adipocytes: comparison of A-14 with A-19 125I-labelled insulin

We compared A-14 and A-19 125I-labelled insulin in receptor-binding and degradation. Percent receptor-binding of A-14 and A-19 125I-labelled insulin to 2.4 X 10(9)/ml erythrocytes after 210 min incubation at 15 degrees C was 7.8 and 4.9%, respectively. Percent insulin-receptor binding of A-14 insulin was 1.6 times greater than that of A-19 insulin. A similar result was obtained in an adipocytes insulin binding study. Percent receptor-binding of A-14 and A-19 insulin to 2 X 10(5)/ml fat cells after 30 min incubation in the above buffer was 3.9 and 2.4%, respectively. Degradation of A-14 and A-19 insulin in rat adipocytes was also studied by molecular sieve column chromatography. Isolated rat adipocytes were allowed to associate with A-14 and A-19 125I-insulin for 60 min at 37 degrees C, pH 8.0 in a HEPES-phosphate buffer, and then cells were separated from the buffer by centrifugation. After solubilization with triton X-100, both the solubilized cells and the incubation medium were applied to the Bio-Gel P-30 column to assess the insulin degradation. Degradation of A-14 125I-insulin by the isolated rat adipocytes was 1.6 times greater than that of A-19 125I-insulin. Furthermore, the peak which was thought to be intermediate degradation products of insulin was obtained between the peak of intact insulin and that of 125I-tyrosine. Such a peak of intermediates was much smaller in the incubation media than in the cell-associated materials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of insulin of the honeybee (Apis mellifica)

In the honeybee (Apis mellifica), insulin-like material was partially purified with acid ethanol extractions by a classic method for recovering insulin and following gel filtration on a Sephadex G-50 column. The preparations were characterized by their ability to cross-react with porcine insulin antibodies. Insulin-like biological activity was demonstrated using the insulin bioassay. Stimulation of glucose oxidation or lipogenesis was measured by isolated rat adipocytes. Insulin seems to be more widespread in invertebrates than was previously assumed.     

Receptor binding and biological potency of despentapeptide insulin

The receptor binding and biological potency of despentapeptide insulin (DPI) was assessed in human adipocytes, rat adipocytes and rat hepatocytes. DPI displayed a lower affinity for binding to both human adipocytes (half-maximum displacement at 0.89 +/- 0.04 and 0.20 +/- 0.02 nmol/l for DPI and insulin respectively; P less than 0.001) and rat adipocytes (half-maximum displacement at 7.12 +/- 1.06 and 1.14 +/- 0.18 nmol/l respectively, P less than 0.05). However, although DPI was less potent than unmodified insulin in stimulating glucose uptake in rat adipocytes (half-maximal stimulation at 2.0 +/- 0.67 and 0.47 +/- 0.18 nmol/l respectively; P less than 0.05), DPI was equipotent with insulin in human adipocytes (half-maximal stimulation at 0.034 +/- 0.001 and 0.027 +/- 0.001 nmol/l respectively; P greater than 0.2). In rat hepatocytes, DPI was twofold less potent in binding displacement activity (half-maximum displacement at 3.8 +/- 0.9 and 1.7 +/- 0.3 nmol/l respectively; P less than 0.01) but appeared to be equivalent in stimulating amino butyric acid uptake (half-maximum stimulation at 0.98 +/- 0.12 and 0.95 +/- 0.26 nmol/l respectively). The difference in affinity of DPI binding to rat liver membranes was less marked (1.3 fold decreased compared with insulin: 5.3 +/- 0.7 and 4.2 +/- 0.6 nmol/l respectively; P less than 0.001). Thus, the decreased receptor affinity of DPI was reflected in decreased biological potency in rat adipocytes, but not in human adipocytes nor rat hepatocytes. These data suggest differences in the binding-action linking in the cells of different tissues and different species.     

Processing of proinsulin by transfected hepatoma (FAO) cells.

Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV retroviral vector). Using the DOL vector, production of insulin immunoreactive material was stimulated up to 30-fold by dexamethasone (5 x 10(-7) M). For both proinsulins, fractional release of immunoreactive material relative to cellular content was high, in keeping with the absence of any storage compartment for secretory proteins in these cells. Pulse-chase experiments showed kinetics of release of newly synthesized products in keeping with release via the constitutive pathway. High performance liquid chromatography analysis showed immunoreactivity in the medium distributed between three peaks. For rat proinsulin II, the first coeluted with intact proinsulin; the second coeluted with des-64,65 split proinsulin (the product of endoproteolytic attack between the insulin A-chain and C-peptide followed by trimming of C-terminal basic residues by carboxypeptidase); the third (and minor peak) coeluted with native (fully processed) insulin. For human proinsulin, by contrast, the second peak coeluted with des-31,32 split proinsulin (split and trimmed at the B-chain/C-peptide junction). Analysis of cellular extracts showed intact proinsulin as the major product. The generation of the putative conversion intermediates and insulin was not due to proteolysis of proinsulin after its release but rather to an intracellular event. The data suggest that proinsulin, normally processed in secretory granules and released via the regulated pathway, may also be processed, albeit less efficiently, by the constitutive pathway conversion machinery. The comparison of the sites preferentially cleaved in rat II or human proinsulin suggests cleavage by endoprotease(s) with a preference for R/KXR/KR as substrate.     

Uptake studies in adipocytes isolated from rainbow trout (Salmo gairdnerii). A comparison with adipocytes from rat and cat

Adipocytes were isolated from mesenteric adipose tissue of rainbow trout (Salmo gairdnerii) by incubation of tissue slices at 20 degrees C in a buffer containing 3 mg collagenase per ml. These cells were compared to adipocytes from the cat and the rat, isolated by conventional technique (1 mg collagenase per ml buffer, incubation temperature 37 degrees C). Uptake studies of some metabolites were performed with fish, rat and in some cases cat adipocytes. At a glucose concentration of 0.33 mM, the glucose uptake into rat cells was more than twice as fast as in cells from the cat, and more than five times as fast as in trout cells. 2-Amino butyrate resembled glucose in relative uptake rates between species. Metabolite uptake into rat cells was specific, with different uptake rates for different metabolites. The uptake into trout adipocytes proceeded at similar rates for all metabolites tested, provided the concentrations were the same. The uptake rate of glucose into rat cells was stimulated by insulin. Insulin had no effect on glucose uptake into adipocytes from trout.     

Biological effects of sulphated insulin in adipocytes and hepatocytes

Summary The binding affinity of sulphated insulin compared with unmodified, neutral insulin has been reported to be approximately four times lower in human and rat adipocytes but over twenty times lower in rat hepatocytes. In the present study the biological action of sulphated insulin was assesed in rat hepatocytes and human and rat adipocytes. To achieve half-maximal stimulation of fatty acid synthesis in rat hepatocytes about twenty one times higher concentrations of sulphated than neutral insulin were required (15.07±5.50 vs 0.71±0.34 nmol/l), this ratio being similar to the ratio of binding affinity in rat hepatocytes. In human adipocytes, half-maximal stimulation of initial rates of glucose uptake was observed at 11.6±5.1 vs 2.9±1.3 pmol/l for sulphated and neutral insulin respectively, and half-maximal inhibition of lipolysis at 31.0±13.5 vs 7.3+2.5 pmol/I respectively. These data are consistent with the four-fold lower binding affinity of sulphated insulin to human adipocytes. However, in rat adipocytes the biological potency of sulphated insulin was found to be much lower than anticipated from the binding data, half-maximal stimulation of initial rates of glucose uptake being observed at 757±299 vs 35±13 pmol/l respectively and half-maximal inhibition of lipolysis at 35.9±12.1 vs 1.5±0.5 pmol/l respectively. Thus, in rat adipocytes, approximately 22 times the concentration of sulphated insulin was required to achieve equivalent biological effect. A discrepancy between binding affinity and biological action with respect to sulphated insulin was identified in rat adipocytes but not human adipocytes nor rat hepatocytes suggesting differences in the binding-action linkage in these cells.     

Structure-function studies of an IGF-I analogue that can be chemically cleaved to a two-chain mini-IGF-I

The structure and biological activities of two disulphide isomers of a C-region deletion mutant of insulin-like growth factor-I (IGF-I) which has an Asn--Gly link engineered at the junction of the A- and B-regions were studied before and after chemical cleavage. Circular dichroism (CD) spectra and binding affinity to IGF binding protein 3 (IGFBP3) indicated that the treatment with hydroxylamine did not disrupt the overall tertiary fold of the hormones. Cleavage restored some binding affinity for the IGF-I receptor in both isomers and weakly restored the ability to stimulate incorporation of tritiated thymidine into DNA in NIH 3T3 fibroblasts transfected with the human IGF-I receptor. Cleavage also restored metabolic capacity, as measured by the ability of the isomers to promote lipogenesis in isolated rat adipocytes through the insulin receptor. These results are consistent with the theory that binding of IGF-I to the IGF-I receptor requires a conformational change similar to that involved in insulin binding the insulin receptor. The weak affinity for the IGF-I receptor after cleavage is consistent with the belief that residues in the C-region interact with the IGF-I receptor. This structural difference between insulin and IGF-I gives each a higher binding affinity for its own receptor.     

Absence of covalent binding by insulin to erythrocyte and reticulocyte insulin receptors

We investigated whether insulin forms covalent bonds with its receptors on erythrocytes and reticulocytes, as it does in adipocytes (1). Of the [125I]-insulin specifically bound at 37 degrees C to human and rat erythrocytes and rat reticulocytes, only 1.5-2.3% was non-dissociable on extensive washing. When ghosts prepared from the washed cells were solubilized in Triton X-100, only 0.6-1.5% of the specifically bound radioactivity appeared in the void volume of a Sephadex G-50 column. Moreover in contrast to adipocytes, this high molecular weight radioactivity was not immunoprecipitable by antibodies to the insulin receptor and was dissociated during chromatography in sodium dodecyl sulphate. Thus we have been unable to demonstrate the formation of covalent bonds between insulin and its receptors on erythrocytes and reticulocytes. This finding is consistent with the hypothesis that covalent binding of insulin is a necessary receptor modification for insulin's metabolic effects.     

Degradation of the four monoiodoinsulin isomers by the insulin protease of rat liver

The cleavage of insulin by the partially purified insulin protease was studied using the four [¹²⁵I]tyrosine-monoiodoinsulins (tyrosine A-14 and A-19 of the A-chain; tyrosine B-16 and B-26 of the B-chain). The rates of conversion of the four isomers to trichloroacetic acid-soluble form was in the order B-26 > A-14 > A-19 > B-16. The following was observed in experiments which gave 19/14/5/3 percent conversion to trichloroacetic acid-soluble products: the loss of ability to bind to IM-9 lymphocytes was approx. 55% for all four isomers. About 70% of the radioactivity was in the ‘insulin’ peak, and about 30% was in peptides smaller than insulin as judged by gel filtration on Sephadex G-50. The descending limb of the ‘insulin’ peak contained significant amounts of radioactive material not binding to IM-9 lymphocytes. This material showed multiple peaks when applied to high performance liquid chromatography. Other experiments were designed to cause an almost complete degradation of the isomers. Under these conditions, the radioactivity eluted on Sephadex G-50 largely as iodotyrosine (and some small peptides) using the A-14, B-16 and B-26 isomers, whereas iodotyrosine was absent using the A-19 isomer. Thus, the insulin protease appears to first degrade insulin to multiple products with molecular sizes slightly smaller than insulin and subsequently to small peptides (e.g. containing tyrosine A-19) and amino acids (e.g. tyrosine A-14, B-16 and B-26).     

Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C-peptide) inhibits proinsulin to insulin conversion by transfected pituitary corticotroph (AtT20) cells

The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.     

Receptor binding and biological potency of several split forms (conversion intermediates) of human proinsulin. Studies in cultured IM-9 lymphocytes and in vivo and in vitro in rats

The biological activities of several derivatives of human proinsulin (HPI) containing peptide bond cleavages or peptide deletions in the connecting peptide region were examined in vivo in rats and in several in vitro systems. The two derivatives which were tested in vivo, split (32-33)HPI and des-(64,65)HPI, both demonstrated greater potency in lowering blood glucose than did intact HPI. The receptor binding affinities of split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, des-(33-56)HPI, des-(31,32)HPI, split (32-33)HPI, and split (56-57)HPI were examined in cultured IM-9 lymphocytes, freshly isolated rat adipocytes, and purified rat liver membranes and were compared to the binding of intact HPI and insulin. In these systems, HPI averaged approximately 1% of the activity of insulin. Modification of proinsulin in the connecting peptide region near the A-chain of insulin to form split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, or des-(33-56)HPI resulted in an increase in affinity for receptor binding ranging from 11 to 27-fold over that of intact HPI. In contrast, modifications near the B-chain of insulin to form either des-(31,32)HPI or split (32-33)HPI resulted in roughly a 5-fold increase in affinity, whereas a cleavage within the connecting peptide to form split (56-57)HPI showed only a 2-fold increase in affinity as compared to intact HPI. The biological potencies of these materials were examined in isolated rat adipocytes. At high concentrations (10(-7) M), each derivative produced the same maximal response. At lower concentrations, differences in the relative potencies paralleled the differences in receptor binding affinity previously noted.     

Regulation of lipoprotein lipase. Induction by insulin.

Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.     

Nature of the inhibitory effect of collagenase on phosphodiesterase activity

The level of phosphodiesterase (PDE) activity is lower in collagenase-isolated human fat cells than in adipose tissue fragments. The inhibition is not species-specific since collagenase also inhibits PDE in rat adipose tissue and bovine heart. In subcellular fractions from isolated fat cells, the PDE activities were lowest in the plasma membrane-enriched fractions and highest in the cytosolic fractions. This is opposite to PDE in subcellular fractions obtained from adipose tissue fragments. In dose-response experiments, collagenase inhibited particulate PDE to a much larger extent than it inhibited soluble PDE. The extracellular activities of PDE were completely eliminated by collagenase. Repeated washings or reincubation of the isolated fat cells did not restore the PDE activity. A purified collagenase with low specific protease activity reduced the PDE activity in isolated fat cells to a lesser extent than did a collagenase with high specific protease activities. Collagen and several protease inhibitors were ineffective in preventing the reduction of PDE after exposure to collagenase. It is concluded that nonspecific proteases in the collagenase preparations used for fat cell isolation interact with particulate and soluble PDE causing an irreversible inhibition of PDE activity in isolated fat cells. Of the various forms of PDE, plasma membrane-associated PDE seems most sensitive to collagenase.     

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

The effects of glucose on insulin biosynthesis were studied by measuring the incorporation of radiolabelled amino acids into proinsulin/insulin in isolated rat islets. The islets were pulse labelled for 15 min with [3H]leucine (present in rat insulin I and II) or [35S]methionine (unique to rat insulin II) and then incubated for a 165 min post-label (chase) period during which the majority of labelled proinsulin was converted to insulin but under conditions whereby greater than 95% of radiolabelled proinsulin or insulin was retained in the islets. The newly synthesized, labelled, insulin was analyzed by high performance liquid chromatography. Rat I and II insulin biosynthesis was stimulated by 16.7 mM glucose to the same extent.