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M Y Zhou

Publications and source records attributed to M Y Zhou.

At least 37 records · Page 2Linked to original sources
PubMed1996

High affinity NAD(+)-dependent 11 beta-hydroxysteroid dehydrogenase in the human heart.

Receptor-ligand binding is an essential component of mineralocorticoid (MC) activity in target tissues. Detection of type 1 mineralocorticoid receptors (MR) in cardiac tissue is therefore suggestive that, like kidney, the heart is MC responsive. The presence of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) within MC responsive tissue is essential to prevent saturation of MR by glucocorticoids. Using both high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC), we have found that a high-affinity species of 11 beta-HSD predominates within human heart. Although two 11 beta-HSD isoforms were detected in human cardiac tissues, the activity of high-affinity (type 2) 11 beta-HSD was found to be at least twice that of low affinity (type 1) 11 beta-HSD. Human cardiac type 2 11 beta-HSD possesses characteristics identical to the high-affinity enzyme of distal renal tubules; 11 beta-dehydrogenation of corticosterone or cortisol to their 11-keto metabolites is NAD(+)-dependent and, with corticosterone as substrate, the enzyme has a nanomolar Km (15.1 nM as determined by Lineweaver-Burke analysis). Furthermore, its activity is unidirectional; corticosterone and cortisol are 11 beta-dehydrogenated to inactive 11-keto metabolites, whereas 11-oxoreductase activity (conversion of 11-dehydrocorticosterone and cortisone to corticosterone and cortisol, respectively) is absent. RT/PCR analysis, using primers complementary to the human renal type 2 11 beta-HSD sequence, demonstrated that the high-affinity species of 11 beta-HSD expressed in human heart is indeed the same enzyme as that produced in the kidney. These findings strongly suggest that, as is the case in the distal portion of the nephron, type 2 11 beta-HSD plays an important role in the human heart to promote glucocorticoid metabolism and to confer MC specificity upon MR.

Base Sequence↗
PubMed1996

Corticosteroid synthesis in the central nervous system.

The possibility that adrenocorticosteroids might be synthesized in the central nervous system was assessed by RT-PCR using primers for the CYP11B1 gene which codes for 11 beta-hydroxylase, the enzyme responsible for corticosterone and cortisol formation in the zona fasciculata, incubation of minces of several areas of the brain with 3H-DOC and measuring steroid metabolites, and determining the effect of the intracerebroventricular infusion of the 11 beta-hydroxylase mechanism-based inhibitor 19-ethynyldeoxycorticosterone upon the salt-induced increase in blood pressure in SS/jr rats. Significant, though small relative to the adrenal, amounts of mRNA for 11 beta-hydroxylase was found in the aorta, cerebrum, cerebellum, hippocampus, hypothalamus and amygdala, but not in the heart. Brain minces converted 3H-DOC to corticosterone and 11-dehydrocorticosterone to a greater degree than to 18-OH-DOC. The effect of 19-ethynyldeoxycorticosterone was dose dependent, with the lower doses preventing salt-induced hypertension and the higher doses having no effect or increasing the blood pressure.

Adrenal Cortex Hormones↗
PubMed1995

Cloning and expression of the rat adrenal cytochrome P-450 11B3 (CYP11B3) enzyme cDNA: preferential 18-hydroxylation over 11 beta-hydroxylation of DOC.

The biosynthesis of glucocorticoids and mineralocorticoids in the rat adrenal cortex requires the action of two different cytochrome P450 11 beta-hydroxylases, CYP11B1 and CYP11B2, which are distributed in the zona fasciculata and glomerulosa, respectively. The existence of another cytochrome P450-11 beta gene, CYP11B3, was recently reported. Although CYP11B3 has similar gene structure and great homology to the CYP11B1 and -B2 genes, the CYP11B3 mRNA was not originally detected by reverse transcription-polymerase chain reaction (RT-PCR) and has only recently been cloned and detected from neonatal rat adrenals. Herein we demonstrate RT-PCR detection of CYP11B3 mRNA expressed in adult rat adrenal and brain tissues. The whole coding region of the CYP11B3 enzyme cDNA was cloned and sequenced. When transiently expressed in COS-7 cells the CYP11B3 converted deoxycorticosterone (DOC) to corticosterone and 18-hydroxydeoxycorticosterone, but not to 18-hydroxycorticosterone or aldosterone. It produced more 18-OH-DOC than corticosterone. A single mutation in CYP11B3 in which Gly-59 was replaced by Ser, reduced the enzymatic activity 5-6-fold. Furthermore, CYP11B3 mRNA expression is greater in neonatal, compared to adult rat adrenal glands.

Adrenal Glands↗
PubMed1995

Postprandial triglyceride and retinyl ester responses to oral fat: effects of fructose.

It has been shown that addition of fructose to an oral fat load results in higher postprandial concentrations of triglyceride. The present study, performed in 11 healthy volunteers, was initiated to see whether the effect of fructose on fat-induced lipemia also involved changes in postprandial concentrations of triglyceride-rich lipoproteins of intestinal origin. Vitamin A was used to label intestinal lipoproteins, and the retinyl palmitate concentrations were determined in plasma and in the Sf > 400 and Sf 20-400 lipoprotein fractions (Sf denotes the Svedberg flotation index). Addition of fructose (50 g) to a standard (40-g oral) fat load resulted in higher postprandial concentrations of triglyceride and retinyl palmitate in plasma and the Sf > 400 lipoprotein fraction (P < 0.001, analysis of variance), and the higher the fasting plasma triglyceride concentration, the greater the magnitude of the fructose effect (r = 0.83, P < 0.002). These data show that triglyceride-rich lipoproteins of intestinal origin play a role in the fructose-induced accentuation of postprandial lipemia.

Administration, Oral↗
PubMed1995

Effect of variations in oral fat and carbohydrate load on postprandial lipemia.

In this study we assessed the acute effects of the consumption of varying amounts of fat and fructose on the magnitude of postprandial lipemia. Subjects were studied after an overnight fast on four separate mornings, ingesting in random order 5, 40, or 80 g fat, or 5 g fat plus 50 g fructose. Vitamin A (36 mg, or 120,000 U retinol) was also given and blood was drawn at frequent intervals over the next 10 h for measurement of triacylglycerol and retinyl palmitate (RP) concentrations in plasma and the Sf > 400 and Sf 20-400 lipoprotein fractions. (Sf denotes flotation units.) In general, the postprandial triacylglycerol response increased in plasma and in both lipoprotein fractions as a function of both the baseline fasting triacylglycerol concentration and the amount of fat ingested. However, no matter how high the fasting plasma triacylglycerol concentration, there was no increase in the postprandial triacylglycerol concentration in plasma or either lipoprotein fraction after the 5-g oral fat load. The results of the measurements of RP concentration were somewhat similar in that there was a dose-dependent increase in the plasma and the Sf > 400 lipoprotein fraction in response to the higher fat loads. However, just the opposite was true in the Sf 20-400 lipoprotein fraction, for which the increase in RP concentration was inversely related to the size of the fat load.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

MRF4, Myf-5, and myogenin mRNAs in the adaptive responses of mature rat muscle.

We studied the possible role of specific muscle regulatory factors (MRF) in the adaptive response to changes in contractile activity in mature skeletal muscle. The tibialis anterior muscle of anesthetized female rats was subjected to low-frequency stimulation, static stretch, or a combination of both. Message levels of MRF were observed after 2 h of activity, and the subsequent 20-h recovery period by slot blot and in situ hybridizations for MRF4, Myf-5, and myogenin. A combination of stimulation and stretch for 2 h increased MRF4 (11.6 +/- 5.3-fold) and Myf-5 (6.6 +/- 1.4-fold). In situ hybridization showed abundance in some regions of the muscle with positive staining near peripheral nuclei of both large and small fibers. Message levels remained high for 30 min and declined to near control levels by 20 h of recovery. Myogenin mRNA levels were unaffected by any manipulations. Neither stretch alone nor 10 Hz of electrical stimulation alone induced a significant increase in MRF. We conclude that myonuclei, and possibly activated myoblasts, increase expression of Myf-5 and MRF4 after a combination of both stimulation and stretch for 2 h.

Adaptation, Physiological↗
PubMed1995

Human fiber size and enzymatic properties after 5 and 11 days of spaceflight.

Biopsies from the vastus lateralis muscle were obtained from three astronauts before and after two 5-day flights and from five astronauts before and after one 11-day flight (space shuttle flights: STS-32, -33, and -34). Muscle fibers from two separate samples from each biopsy were classified as type I and II or as type I, IIA, and IIB by using qualitative myofibrillar adenosinetriphosphatase (ATPase) staining. Cross-sectional area (CSA), number of capillaries per fiber, and the activities of succinate dehydrogenase (SDH), alpha-glycerophosphate dehydrogenase (GPD), and myofibrillar ATPase were determined from one sample of fibers of each myofibrillar ATPase type. Postflight biopsies had 6-8% fewer type I fibers than preflight. Mean fiber CSAs were 16-36% smaller after the 11-day flight with the relative effect being type IIB > IIA > I. Mean fiber CSAs were 11 and 24% smaller in type I and II fibers after 5 days of flight. Myofibrillar ATPase activities increased in type II but not in type I fibers after flight, whereas SDH activity was unaffected in either fast or slow fibers. GPD activity in type I fibers was approximately 80% higher (P > 0.05) postflight compared with preflight. Myofibrillar ATPase/SDH ratios in type II fibers were higher after than before flight, suggesting that some fast fibers were more susceptible to fatigue after flight. The GPD/SDH ratios were elevated in some type I fibers after spaceflight. The number of capillaries per fiber was 24% lower after than before flight, whereas the number of capillaries per unit CSA of muscle tissue was unchanged. These data suggest that adaptations in the size, metabolic properties, and vascularity of muscle fibers can occur rapidly in the space environment. These adaptations were qualitatively similar to those observed in animals after actual or simulated spaceflight conditions for short periods.

Adenosine Triphosphatases↗
PubMed1995

Myosin heavy chain isoforms of human muscle after short-term spaceflight.

The influence of microgravity on the myosin phenotype of skeletal muscle fibers in the vastus lateralis of eight crew members was studied before and after 5-day (n = 3) and 11-day (n = 5) spaceflights (space shuttle flights: STS-32, -33 and -34). Single-fiber electrophoresis analyses showed that the proportion of fibers expressing only slow (type I) myosin heavy chain (MHC) in the vastus lateralis was significantly lower after than before 11 days of spaceflight. Although the family of type II MHC isoforms was elevated post- compared with preflight, the distribution among the isoforms of type II MHC was not statistically different. Based on monoclonal and polyclonal antibodies specific for three adult MHC isoforms and single-fiber electrophoresis, approximately 3% of the fibers analyzed coexpressed all three adult MHC isoforms. The results from immunohistochemical staining with two different sets of antibodies indicate a reduction in the percentage of fibers expressing type I MHC as a result of spaceflight. The mean difference, however, was significant only when the fibers were categorized simply as type I or II. These changes appeared to be highly individualized among the astronauts. These results suggest that a rapid change in MHC isoform expression can occur in some muscle fibers after a relatively brief exposure to spaceflight.

Adult↗
PubMed1995

Relation between insulin resistance, hyperinsulinemia, postheparin plasma lipoprotein lipase activity, and postprandial lipemia.

We examined the relation between insulin resistance, plasma glucose and insulin responses to meals, lipoprotein lipase (LPL) activity, and postprandial lipemia in a population of 37 healthy nondiabetic individuals. Plasma glucose and insulin concentrations were determined at frequent intervals from 8 AM through midnight (breakfast at 8 AM and lunch at noon); resistance to insulin-mediated glucose disposal was determined by measuring the steady-state plasma glucose (SSPG) concentration at the end of a 180-minute infusion of glucose, insulin, and somatostatin; LPL activity was quantified in postheparin plasma; and postprandial concentrations of triglyceride (TG)-rich lipoproteins were assessed by measuring the TG and retinyl palmitate content in plasma and the Svedberg flotation index (Sf) > 400 and Sf 20 to 400 lipoprotein fractions. Significant simple correlation coefficients were found between various estimates of postprandial lipemia and SSPG (r = .38 to .68), daylong insulin response (r = .37 to .58), daylong glucose response (r = .10 to .39), and LPL activity (r = -.08 to -.58). However, when multiple regression analysis was performed, only SSPG remained independently associated with both postprandial TG and retinyl palmitate concentrations. These data provide evidence that insulin resistance plays an important role in regulating the postprandial concentration of TG-rich lipoproteins, including those of intestinal origin.

Adult↗
PubMed1995

Cloning, expression, and tissue distribution of the rat nicotinamide adenine dinucleotide-dependent 11 beta-hydroxysteroid dehydrogenase.

A pcDNAI adult rat kidney complementary DNA (cDNA) library was screened using a sheep 11-hydroxysteroid dehydrogenase 2 (11 beta HSD-2) probe, and the isolated clones were sequenced. The 5'-end of the cDNA was determined by 5'-rapid amplification of cDNA ends. The rat 11 beta HSD-2 cDNA is 1864 base pair (bp) long. It consists of a 5'-untranslated region of 126 bp, an open reading frame of 1203 bp, and a 3'-untranslated region of 535 bp. The predicted protein contains 400 amino acid residues, with a calculated mol wt of 43,700. The rat 11 beta HSD-2 protein sequence is 85% homologous to human 11 beta HSD-2 and 76% to sheep 11 beta HSD-2. Expression of 11 beta HSD-2 messenger RNA by Northern blot and reverse transcription-polymerase chain reaction was high in kidney, distal colon, and adrenal and lower in the lung, hypothalamus, hippocampus, and midbrain. The rat 11 beta HSD-2 was transiently transfected into modified Chinese hamster ovary cells. Cells transfected with the 11 beta HSD-2 cDNA converted corticosterone into 11-dehydrocorticosterone. Conversion of corticosterone to 11-dehydrocorticosterone was NAD+ dependent and had a Km of 10.1 +/- 2.1 nM. In conclusion, we have cloned a rat NAD(+)-dependent 11 beta HSD with tissue distribution and kinetic characteristics suggesting that it could play a significant role in mineralocorticoid receptor selectivity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Why do low-fat high-carbohydrate diets accentuate postprandial lipemia in patients with NIDDM?

OBJECTIVE: To understand why low-fat high-carbohydrate (CHO) diets lead to higher fasting and postprandial concentrations of triglyceride (TG)-rich lipoproteins in patients with non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: Patients with NIDDM were placed randomly on diets containing either 55% CHO, 30% fat, and 15% protein or 40% CHO, 45% fat, and 15% protein for 6 weeks, followed by crossover to the other diet. Test meals at the end of each diet period were consumed at 8:00 A.M. and 12:00 P.M. (noon) and contained 20 and 40% of daily calories, respectively. Vitamin A was also given at noon, and TG-rich lipoproteins of intestinal origin were identified by the presence of vitamin A esters. Frequent measurements were made throughout the 24-h study period of plasma glucose, insulin, and TG concentrations. Plasma samples obtained from 12:00 P.M. (noon) until 12 A.M. (midnight) were subjected to ultracentrifugation, and measurements were made of TG and vitamin A ester concentrations in plasma and in both the Svedberg flotation constant (Sf) > 400 (chylomicron) and Sf 20-400 (chylomicron remnant) lipoprotein fractions. In addition, very-low-density lipoprotein (VLDL)-TG turnover rate was estimated by following the decay of [3H]VLDL-TG. Finally, postheparin lipoprotein lipase and hepatic lipase activities were measured at the end of each dietary period. RESULTS: Mean +/- SE hourly concentrations of glucose (8.0 +/- 0.8 vs. 7.5 +/- 0.7 mmol/l), insulin (184 +/- 26 vs. 158 +/- 19 pmol/l), and TG (2.8 +/- 0.2 vs. 2.1 +/- 0.2 mmol/l) were higher (P < 0.05-0.001) after the 55% CHO diet. The 55% CHO diet also led to an increase (P < 0.05-0.01) in the mean +/- SE hourly concentrations of vitamin A esters in plasma (2.3 +/- 0.3 vs. 1.6 +/- 0.1 mumol/l) and in both the chylomicron (2.0 +/- 0.3 vs. 1.4 +/- 0.1 mumol/l) and chylomicron remnant fractions (0.36 +/- 0.04 vs. 0.14 +/- 0.03 mumol/l). In addition, the VLDL-TG production rate was higher (17.2 +/- 1.4 vs. 12.8 +/- 1.0 mg.kg-1.h-1, P < 0.003) and the VLDL-TG fractional catabolic rate lower (0.22 +/- 0.02 to 0.28 +/- 0.02 l/h, P < 0.005) after the 55% CHO diet. Finally, there was an increase in lipoprotein lipase activity (7.0 +/- 0.8 to 8.1 +/- 0.7 mumol free fatty acids released .ml-1.h-1, P < 0.02) in response to the CHO-enriched diet. CONCLUSIONS: A low-fat high-CHO diet in patients with NIDDM led to 1) higher day-long plasma glucose, insulin, and TG concentrations; 2) postprandial accumulation of TG-rich lipoproteins of intestinal origin; 3) increased production of VLDL-TG; and 4) increased postheparin lipoprotein lipase activity. These data provide a mechanism for the hypertriglyceridemic effect of CHO-enriched diets in patients with NIDDM and demonstrate that multiple risk factors for coronary heart disease are accentuated when these individuals consume diets recommended to reduce this risk.

Adult↗
PubMed1995

[Intercellular relationship of notochord determination of Xenopus laevis].

During the process of determination, the presumptive notochord is situated beneath neuroepithelium, flanked at two sides by presumptive somites and underlain with archenteron roof ventrally. Among these neighbouring embryonic tissues, presumptive somites were found to exert the main influence on notochord determination. By electron microscopic observations, the presumptive notochord and somite cells were seen to situate either close to each other (plate I, Fig. 1) or connected by cytoplasmic processes forming intercellular lumen (plate I, Fig. 5). Coated pits and coated vesicles appeared at the outer surface of both types of cells (plate I, Figs. 1-4). For the presumptive somite cells, spherical bodies of different sizes and variable contents were observed either near or protruding from the outer surface (plate II, Figs. 6-10). The spherical bodies were also found in the intercellular lumen (plate III, Fig. 11). These spherical bodies were mainly composed of granules, loosely scattered or densely packed. The granules were of similar size and similar shade of electron staining as those of ribosomes of the presumptive somite cells. For the presumptive notochord cells, no spherical bodies of the above mentioned type were found, but phenomenon of engulfing luminal material was observed (plate III, Fig. 12). The significance of the appearance of these spherical bodies in the determination of notochord cells has been discussed.

Animals↗
PubMed1994

The hybrid rat cytochrome P450 containing the first 5 exons of the CYP11B1 and last 4 exons from the CYP11B2 enzyme retains 11 beta-hydroxylase activity, but the alternative hybrid is inactive.

Human, mouse and rats have 2 different cytochrome P-450 11 beta-hydroxylases in the adrenal cortex. The classical rat 11 beta-hydroxylase or CYP11B1 enzyme hydroxylates deoxycorticosterone to corticosterone and 18-hydroxydeoxycorticosterone and is located throughout the adrenal. The second aldosterone synthase or CYP11B2 enzyme is located in the zona glomerulosa and converts deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. In rat the coding nucleotide sequence and the deduced amino acid sequences of the CYP11B1 and CYP11B2 genes are homologous by 88% and 83%, respectively. We have constructed two different hybrid cDNAs by exchanging two fragments of the rat CYP11B1 and CYP11B2 at the junction of the 5/6 exon and expressed them in COS 7 cells. The hybrid CYPH11B1 construct containing the first 5 exons of the CYP11B1 when expressed, retains 11 beta-hydroxylase activity, but cannot process corticosterone to 18-hydroxycorticosterone or aldosterone. The hybrid CYPH11B2 construct containing the first 5 exons of the CYP11B2 enzyme when expressed is inactive.

Animals↗
PubMed1994

Effect of glipizide treatment on postprandial lipaemia in patients with NIDDM.

The primary goal of the present study was to examine the effects of improved glycaemic control associated with glipizide treatment on postprandial lipaemia in non-insulin-dependent diabetic patients. The metabolism of triglyceride-rich lipoproteins of intestinal origin was assessed by measuring the retinyl palmitate content in plasma and the Svedberg flotation index (Sf) > 400 and Sf 20-400 lipoprotein fractions. Fasting plasma glucose concentrations (14.5 +/- 0.5 vs 9.0 +/- 0.5 mmol/l), glycated haemoglobin levels (13.1 +/- 0.6 vs. 9.7 +/- 0.6%), and daylong plasma glucose concentrations were all significantly lower after glipizide treatment (p < 0.001). The improvement in glycaemic control was associated with increases in insulin-mediated glucose uptake (p < 0.001) and plasma post-heparin lipoprotein and hepatic lipolytic activities (p < 0.02). Both fasting plasma triglyceride (3.09 +/- 0.51 vs 2.37 +/- 0.34 mmol/l), and postprandial triglyceride concentrations (p < 0.05-0.001) were lower following glipizide treatment, associated with a significant fall in retinyl palmitate content in all three lipoprotein fractions (p < 0.02-0.001), with the most substantial decrease seen in the Sf20-400 fraction. These data indicate that glipizide-induced improvement in glycaemic control was associated with changes in the metabolism of triglyceride-rich lipoproteins of intestinal origin that would be anticipated to reduce risk of coronary heart disease in non-insulin-dependent diabetic patients.

Blood Glucose↗
PubMed1994

Effect of metformin on postprandial lipemia in patients with fairly to poorly controlled NIDDM.

OBJECTIVE: To quantify the effect of metformin on the metabolism of triglyceride (TG)-rich lipoprotein of intestinal origin in patients with non-insulin-dependent diabetes mellitus (NIDDM) who had responded to sulfonylurea but still had fasting hyperglycemia. RESEARCH DESIGN AND METHODS: Sixteen patients with NIDDM who had demonstrated a fall in fasting plasma glucose concentration > 2.2 mmol/l in response to glipizide treatment but continued to have fasting plasma glucose concentrations > 8.3 mmol/l were studied. Fasting glucose, GHb, lipid and lipoprotein concentrations were determined, and resistance to insulin-mediated glucose disposal was estimated by measuring the steady-state plasma glucose (SSPG) concentration at the end of a 180-min infusion of somatostatin, glucose, and insulin. In addition, plasma glucose, insulin, and TG concentrations were measured at frequent intervals from 0800 to 2400, with patients eating breakfast at 0800 and lunch at 1200. Vitamin A was also given at lunch, and the retinyl ester content in plasma and in chylomicron (Svedberg flotation constant [Sf] > 400) and the chylomicron remnant (Sf 20-400) fractions were used to quantify the concentration of postprandial intestinal TG-rich lipoprotein from 1200 to 2400. RESULTS: Fasting plasma glucose concentrations (6.8 +/- 0.4 vs. 10.5 +/- 0.4 mmol/l), GHb levels (7.9 +/- 0.3 vs. 10.8 +/- 0.5%), and day-long plasma glucose concentrations were all significantly lower after metformin treatment (P < 0.001), which was associated with a significant (P < 0.001) fall in SSPG concentration (11.0 +/- 0.9 to 9.6 +/- 0.6 mmol/l). In addition, postprandial concentrations of glucose, insulin, free fatty acids, and TG were lower (P < 0.001) following metformin treatment. Postprandial retinyl ester concentrations were also lower in plasma by 33 +/- 5.7% (P < 0.001) and in both the chylomicron (32 +/- 7.2%, P < 0.001) and chylomicron remnant (26 +/- 7.0%, P < 0.005) fractions. CONCLUSIONS: Addition of metformin to sulfonylurea-treated patients with NIDDM with less than optimal glycemic control was associated with improved glycemic control, lower postprandial insulin and TG concentrations, and a decrease in postprandial concentration of TG-rich lipoproteins of intestinal origin. All of these changes might be expected to decrease risk of coronary heart disease.

Blood Glucose↗
PubMed1992

[Studies on genetic engineering of human insulin-purification and characterization of human proinsulin and insulin].

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.

Cloning, Molecular↗